De novo binding domain containing polypeptides and uses thereof

ABSTRACT

Provided herein are de novo binding domain containing polypeptides (DBDpp) that specifically bind a target of interest. Nucleic acids encoding the DBDpp, and vectors and host cells containing the nucleic acids are also provided. Libraries of DBDpp, methods of producing and screening such libraries and the DBDpp identified from such libraries and screens are also encompassed. Methods of making and using the DBDpp are additionally provided. Such uses include, without limitation, affinity purification, and diagnostic and therapeutic applications.

CROSS-REFERENCE TO RELATED CASES

This application is a continuation of U.S. application Ser. No.15/564,325, 371(c) date Oct. 4, 2017, which is a U.S. National Phase ofPCT Application No. PCT/US2016/025868, filed Apr. 4, 2016, which claimspriority to U.S. Provisional Application Ser. No. 62/143,772, filed Apr.6, 2015, the entirety of each of which is incorporated by referenceherein. All references, patents and patent applications referred toherein are herein incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

The present application is being filed accompanied by a Sequence Listingin electronic format. The Sequence Listing is provided as a fileentitled 6666_0048_Sequence_Listing.txt, created Mar. 12, 2020, andwhich is 147 kilobytes in size. The information in the Sequence Listingis incorporated herein by reference in its entirety.

BACKGROUND

Antibody-based reagents have accelerated the pace of biological researchand development. Antibody compositions represent one of the mostimportant and successful classes of therapeutic and diagnostic agentsutilized in the pharmaceutical industry. However, cost, time andefficacy have motivated the development of alternative affinityreagents.

A variety of non-antibody binding formats have emerged for applicationshistorically served by antibodies. While many successes have beenreported for unstructured, linear peptides, more robust results havebeen achieved by imposing a structural constraint on the peptidesequence—typically through the introduction of a disulfide bond. Thisconstraint affords higher affinity and greater specificity through themore favorable thermodynamics of fixed-shape complementarity and surfacepresentations of residues (e.g., hydrophobic amino acids) that mightotherwise be buried and therefore not target-facing (Ladner, Trends inBiotech. 13(10):426-430, 1995). Conversely, formats that containdisulfide bonds are typically prone to improper pairing of cysteines,either intra-domain or inter-domain, that can lead to lower expression,product yield and product quality.

Structure found in protein subdomains has provided another source ofstructural constraint. Structures such as fibronectin type III repeats(adnectins), z-proteins (affibodies), knottins, lipocalins (anticalins)and ankyrin repeats (DARPins) have been developed with antibody-likeaffinities against a variety of different targets (Hey et al., Trends inBiotech. 23(10):514-422, 2005). These domains typically contain twofeatures that are analogous to the frameworks and complementaritydetermining regions (CDRs) found in antibody variable domains: astructural scaffold that imparts high thermodynamic stability andresidues or loops that form the basis of the display library'svariability.

SUMMARY

In general, there remains a substantial unmet need for newtarget-binding agents and compositions, and particularly for such agentscontaining alternative binding scaffolds (e.g., non-antibody scaffolds).In several embodiments, agents of particular interest may becharacterized by, for example, substantially reduced production costsand/or comparable or superior reagent, diagnostic and/or therapeuticproperties as compared to antibodies. The present disclosure providessuch desirable agents in several embodiments. For example, in severalembodiments, the present disclosure provides certain polypeptide agentsthat are characterized by high target binding affinity and by anon-antibody structural scaffold. Alternatively or additionally, inseveral embodiments, target-binding agents, such as the polypeptidesdisclosed herein, for example resulting from the production methodsdisclosed herein have advantages including, for example, highlytarget-specific binding. In some embodiments, this can advantageously beused to target therapeutics (e.g., immune cells) to particular cells(e.g., diseased cells), thereby reducing or eliminating off-targeteffects. In some embodiments, the agents provided herein, such as thetarget-specific polypeptides, can be used as protein therapeutics tobind cells or soluble factors involved in disease. In some embodiments,the provided agents can be used to purify targets (e.g., proteins orother targets) with a high degree of specificity, which may, forexample, result in higher purity and/or reduced downstream processing topurify a target.

Several embodiments of the inventions disclosed herein relate to agentsthat specifically bind targets of interest, such as the de novo bindingdomain (DBD) containing polypeptides (DBDpp) disclosed herein. Nucleicacids encoding the DBDpp and vectors and host cells containing thenucleic acids are also provided, as are DBDpp libraries and methods forproducing and screening such libraries and the DBDpp identified fromsuch libraries and/or screens. DBDpp including DBDpp fusion proteins arealso provided, as are methods of making and using the DBDpp.Non-limiting examples of such uses include, but are not limited to,affinity purification, target analysis, diagnostic and/or therapeuticapplications.

In several embodiments, there is provided a binding agent that bindswith a high degree of specificity to a target of interest. In severalembodiments the binding agent is a non-antibody agent. In severalembodiments, the binding agent is a polypeptide. In several embodiments,there are provided polypeptides for binding a target of interest thathave a sequence that differs, at least at one position, from thesequence of SEQ ID NO:1. In several such embodiments, the agent (e.g., apolypeptide) exhibits specific binding to the target of interest, thatbinding being greater than the binding of a polypeptide according to SEQID NO:1 to the target of interest. In several embodiments, there isprovided a polypeptide for binding a target of interest, the polypeptidecomprising an amino acid sequence comprisingMGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN(SEQ ID NO:4), wherein the sequence differs in sequence from thesequence of SEQ ID NO:1 (e.g., by modifications to the amino acidsequence of SEQ ID NO:1). In several embodiments, the polypeptidespecifically binds a target of interest (such as a cancer marker orother distinctive marker related to a target of interest), and thespecific binding of the polypeptide to the target of interest is greaterthan binding of a polypeptide according to SEQ ID NO:1 to the target ofinterest. In several embodiments, the polypeptide does not contain thesequence of SEQ ID NO:50.

In several embodiments, the polypeptide has a sequence that differs fromSEQ ID NO:1 because certain selected amino acid positions have beenmodified. In some embodiments, the modifications comprise substitutions.In several embodiments, the substitutions are conservativesubstitutions, while in some embodiments, the substitutions arenon-conservative substitutions. In still additional embodiments,combinations of conservative and non-conservative substitutions areused. In some embodiments, the substitutions do not include substitutionwith a cysteine (e.g., no cysteines are added to the sequence). In someembodiments, wherein the substitutions do not include substitution witha proline (e.g., no prolines are added to the sequence). In someembodiments, neither cysteine nor proline is substituted into thesequence of the polypeptide.

Various targets of interest can be bound by the agents disclosed herein.For example, in several embodiments, the target of interest specificallybound by the polypeptide is a cancer antigen. In some embodiments, thecancer antigen specifically bound by polypeptide is PD-L1. In severalsuch embodiments, target-binding polypeptide comprises or consistsessentially of an amino acid sequence selected from the group consistingof SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,SEQ ID NO: 43, SEQ ID and NO:44. In some embodiments, the cancer antigenspecifically bound by polypeptide is CD137. In some such embodiment, thepolypeptide comprises or consists essentially of an amino acid sequenceselected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18, andSEQ ID NO:19. In some embodiments, the cancer antigen specifically boundby polypeptide is CD123. In some such embodiments, the polypeptidecomprises or consists essentially of an amino acid sequence selectedfrom SEQ ID NOS: 92-127. In some embodiments, a combination of cancerantigens is targeted, for example by coupling or otherwise combiningvarious target-binding polypeptides. In some embodiments, two, three,four or more different cancer antigens are targeted. In someembodiments, multiple target-binding polypeptides are used to enhancethe ability and/or capacity to bind a single target (e.g., dimers,trimers, etc.)

Additionally provided for in several embodiments is a method fortransforming a reference polypeptide into a polypeptide having specificbinding for a target of interest, the method comprising modifying aplurality of amino acid residues from a reference polypeptide togenerate a plurality of candidate binding polypeptides, packaging theplurality of candidate binding polypeptides in a plurality of vectors togenerate a candidate library, and screening the candidate library forcandidate binding polypeptides that exhibit specific binding to thetarget of interest. In several embodiments, the reference polypeptidecomprises a variant of a non-naturally occurring polypeptide andcomprises three anti-parallel alpha helices joined by linker peptides.In several embodiments the amino acid residues to be modified aresolvent accessible or solvent inaccessible amino acids. In severalembodiments, a greater degree of solvent accessible amino acids aremodified, while in some embodiments a greater degree of solventinaccessible amino acids are modified. In some embodiments, themodification comprises amino acid substitutions. As discussed above, thesubstitutions can comprise conservative amino acid substitutions,non-conservative amino acid substitutions, and/or combinations thereof.Optionally, in several embodiments, the substitution does not comprisesubstitution in of a cysteine, does not comprise substitution in of aproline, and in some cases does not comprise substitution in of acysteine or a proline.

In several embodiments, the method further comprises identifyingpotentially immunogenic amino acid residues in the candidate bindingpolypeptides and modifying at least one of the potentially immunogenicamino acid residues (e.g., to reduce the potential immunogenicity of theresultant polypeptides that bind a target of interest). In severalembodiments, the modification to reduce immunogenicity comprises anamino acid substitution (e.g., conservative and/or non-conservativesubstitutions).

In several embodiments, there is provided a de novo binding domainpolypeptide (DBDpp) that comprises or consists essentially of threeanti-parallel alpha helices, the DBDpp being a variant of a syntheticpolypeptide, wherein the DBDpp immunospecifically binds to a proteinthat is at least 95% identical to CD123. In several embodiments, theDBDpp has a dissociation constant (KD) between about 10⁻⁴M and about10⁻¹²M. In some embodiments, the target to which the DBDppimmunospecifically binds comprises amino acids 19-305 of CD123 (SEQ IDNO: 187). There is also provided herein a DBDpp having an amino acidsequenceMGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN(SEQ ID NO:4), and wherein X_(n) is a natural or non-natural amino acid.Moreover, there is also provided for a DBDpp having an amino acidsequence at least 85% identical to the amino acid sequence of any one ofSEQ ID NO:60-SEQ ID NO: 136. Still further embodiments provide for afusion protein that binds to CD123 (or other target of interestdisclosed herein) and further comprises one or more additional DBDppexhibiting binding specificity for a tumor target.

In several embodiments, the target-binding agent (e.g., a polypeptidewith specificity for a target of interest) is labeled. Depending on theembodiment, various labels can be used, including but not limited to anenzymatic label, a fluorescent label, a luminescent label, and abioluminescent label. In some embodiments, the label is a biotin moiety.In several embodiments, a streptavidin moiety can be used. In someembodiments, a His-tag, FLAG-tag or other tag is used. In someembodiments, the label is luciferase, green fluorescent protein, redfluorescent protein, or other similar agent.

In several embodiments, the target-binding agent (e.g., a polypeptide)is conjugated to a therapeutic or cytotoxic agent (e.g.,chemotherapeutic agent, radiotherapeutic agent, etc.). Depending on theembodiment, the target-binding agent may optionally comprise apharmaceutically acceptable carrier.

In several embodiments, there are provided kits comprising any of thetarget-binding agents disclosed herein (e.g., a therapeutic kit, adiagnostic kit, a kit for research use, etc.).

Several embodiments also provide for isolated nucleic acid moleculesencoding the any of the target-binding polypeptides disclosed herein.Still additional embodiments provide for a vector (e.g., a plasmid,viral vector, or non-viral vector) containing the isolated nucleic acidmolecule. Several such embodiments may also include standard componentsfor expression of protein encoded by the nucleic acid (e.g., promoters,packaging components, etc.). For example, in several embodiments, thevector further comprises an additional nucleotide sequence whichregulates the expression of the polypeptide encoded by the nucleic acidmolecule. In several embodiments, the additional nucleic acid sequenceis an inducible promoter.

Further provided for in several embodiments are host cells that comprisethe nucleic acid molecules encoding the any of the target-bindingpolypeptides disclosed herein. In several embodiments such embodiments,the host cell (e.g., a cell line) is engineered to express thetarget-binding polypeptides disclosed herein. In some embodiments, theexpression of the target-binding polypeptides by the host cells allowsproduction and isolation of the target-binding polypeptides. In someembodiments, the expression results in the target-binding polypeptidesexpressed on the surface and/or integral to the membrane of the cells.

Also provided for herein are de novo binding domain polypeptides (DBDpp)that compete with the polypeptides disclosed herein for binding to CD123(or other targets of interest). In several embodiments, there are alsoprovided polypeptides that compete with those disclosed herein forbinding to other targets of interest, including CD123, PD-L1, CD19,CD22, and the like (or other targets disclosed herein). Competitors thatare provided for include full or partial agonists, full or partialantagonists, and the like. Those agents that compete for binding to atarget of interest (either to the same epitope, an overlapping epitope,or a non-overlapping epitope that leads to steric or other hindrances tothe agent binding a target of interest) can be identified by competitivebinding assays.

Also provided for herein are polypeptides (either alone or expressed bya cell) that bind to a tumor. In several embodiments, the binding isbased on the polypeptide having been generated and identified as havingspecific binding for one or more markers expressed by the tumor. Thetumor, depending on the embodiment, may be a suspension tumor or a solidtumor.

Several embodiments, also provide for a chimeric antigen receptor (CAR),wherein the CAR includes a targeting domain, a transmembrane domain, andan intracellular signaling domain. In several embodiments, the targetingdomain is made up of, at least in part, a target-binding polypeptide asdisclosed herein. In several embodiments, the intracellular signalingdomain is selected from the group consisting of a human CD3 zeta domain,41BB domain, a CD28 domain and any combination thereof. Depending on theembodiment, the costimulatory signaling region comprises theintracellular domain of a costimulatory molecule selected from the groupconsisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, and any combination thereof.In several embodiments, the CAR comprises a fusion protein that includesan additional target-binding polypeptide. Also provided for are isolatednucleic acid sequences encoding CARs that include the target-bindingpolypeptides as part (or all) of the targeting region.

Further provided for herein are cells comprising a nucleic acid sequenceencoding a CAR, wherein the CAR comprises an antigen binding domain madeup of, at least in part, a polypeptide that binds a target of interest,a transmembrane domain, and a signaling domain. In several embodiments,the polypeptide binds specifically to a tumor antigen (and thusfunctions to deliver the cell expressing the CAR to the tumor. Inseveral embodiments, the tumor antigen is associated with a hematologicmalignancy. In additional embodiments, tumor antigen is associated witha solid tumor. Both solid and hematologic tumors can be simultaneouslytargeted in some embodiments. In several embodiments, the tumor antigenis selected from the group consisting of CD137, PD-L1, CD123, CTLA4,CD47, KIR, DR5, TIM3, PD1, EGFR, TCR, CD19, CD20, CD22, ROR 1,mesothelin, CD33/1L3Ra, cMet, PSMA, Glycolipid F77, EGFRvIII, GD2,NY-ESO-1, MAGE A3, and combinations thereof. Depending on theembodiment, the cell expressing the CAR can be a T cell or a naturalkiller (NK) cell. In several embodiments, the cell (whether T cell, NKcell or other cell type) exhibits an anti-tumor immunity when thepolypeptide binds to its corresponding tumor antigen.

Still additional embodiments provide for amino acids having the sequenceof SEQ ID 4, wherein X_(n) is not cysteine or proline.

Also provided for in several embodiments are mammalian cells thatgenerate membrane-bound virus-like particles (VLPs), wherein themammalian cell is engineered to express a fusion protein comprising a denovo binding domain polypeptide (DBDpp) fused to a chimeric antigenreceptor (CAR), the fusion protein being expressed on the generated VLPs(e.g., as transmembrane proteins). Depending on the embodiments, theVLPs produced by the mammalian cells are suitable for use as immunogensfor antibody generation. In some such embodiments, the antibodies aredirected against the de novo binding domain polypeptide (DBDpp) (e.g.,the antibodies bind to the DBDpp and can be used to detect the DBDpp,isolate the DBDpp, etc.

The target-binding polypeptides disclosed herein are also useful in atherapeutic context, e.g., for treatment and/or diagnosis of a disease,such as a cancer (e.g., a solid or hematologic malignancy). Thus, thereare provided, in several embodiments methods of treating a subjecthaving cancer, comprising administering to the subject an immune cellcomprising a chimeric antigen receptor (CAR), wherein the CAR comprisesa target binding domain, wherein the target binding domain comprises apolypeptide having an amino acid sequence comprising:MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN(SEQ ID NO:4), a transmembrane domain, and an intracellular domain(comprising a signaling domain). Upon administration to a subject havingcancer, the target binding domain specifically binds to a target ofinterest expressed by a cancer cell, and the binding of the target ofinterest induces the immune cell to generate cytotoxic signals thatresult in cytotoxic effects on the cancer cell, thereby treating thecancer. In several embodiments, the polypeptide has a sequence thatdiffers from SEQ ID NO:1 (e.g., the polypeptide is generated bymodifying the amino acid sequence of SEQ ID NO:1). As a result of thediffering sequence, the polypeptide's specific binding to the target ofinterest is greater than binding of a polypeptide according to SEQ IDNO:1 to the target of interest.

Depending on the embodiment, the immune cell can be a T cell. In someembodiments, the immune cell is a NK cell. Other immune cells, and/orcombinations of different immune cell types can optionally be used. Insome embodiments, combinations of cell types (e.g., NK cells and Tcells) are advantageous because they act synergistically to treat acancer. When combinations are used, the various cell types can targetthe same or different (or overlapping) tumor antigens.

In several embodiments wherein T cells are used, the binding of thetarget of interest stimulates the T cell to initiate intracellularsignaling, produce cytokines, and degranulate, leading to the cytotoxiceffects on the cancer cell. Additionally, in several embodiments, the Tcell proliferates in response to binding the target of interest.Advantageously, however, the activity of the T cell does not result inthe T cells exhibiting a phenotype associated with T cell exhaustion. Inseveral embodiments where T cells are used, the transmembrane domain ofthe CAR comprises 41BB or CD28, and the cytoplasmic domain comprises analpha, beta, or zeta chain of the T cell receptor.

In several embodiments where NK cells are used, the transmembrane domaincomprises CD28, and the cytoplasmic domain comprises a zeta chain of theT cell receptor.

In several embodiments, the CAR-containing immune cells are designed tobind to a target of interest expressed by the cancer cell, such as atumor antigen selected from the group consisting of CD137, PD-L1, CD123,CTLA4, CD47, KIR, DR5, TIM3, PD1, EGFR, TCR, CD19, CD20, CD22, ROR 1,mesothelin, CD33/1L3Ra, cMet, PSMA, Glycolipid F77, EGFRvIII, GD2,NY-ESO-1, MAGE A3, and combinations thereof.

In several embodiments, the CAR further comprises a second polypeptidehaving an amino acid of SEQ ID NO:4, the polypeptide being able tospecifically bind a second target of interest expressed by a cancercell, and wherein the second polypeptide's specific binding the secondtarget of interest is greater than binding of a polypeptide according toSEQ ID NO:1 to the second target of interest. In several embodiments,the generation of the polypeptide that makes up at least a portion ofthe targeting domain of the CAR does not include substituting a cysteineor a proline into SEQ ID NO: 1.

In several embodiments, the administration of the immune cells with aCAR is intravenous, though other routes, such as intra-arterial,intramuscular, local, or other acceptable route can be used for a giventreatment scenario.

There are also provided, in several embodiments, methods of treating asubject having cancer, comprising, administering to the subject animmune cell comprising a chimeric antigen receptor (CAR), wherein theCAR comprises a target binding domain, wherein the target binding domaincomprises a polypeptide having an amino acid sequence selected from ofSEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6,wherein no cysteine or proline residues are substituted into any of SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, whereinthe polypeptide specifically binds a target of interest expressed by acancer cell, and wherein the polypeptide's specific binding to thetarget of interest is greater than binding of a polypeptide according toSEQ ID NO:1 to the target of interest, a transmembrane domain, and anintracellular domain, wherein the intracellular domain comprises asignaling domain, wherein, upon administration to a subject havingcancer, the target binding domain specifically binds to the target ofinterest expressed by a cancer cell, and wherein the binding of thetarget of interest induces the immune cell to generate cytotoxic signalsthat result in cytotoxic effects on the cancer cell, thereby treatingthe cancer. As discussed above, depending on the embodiment, the immunecell can be a T cell, a NK cell, or other type of immune cell (orcombinations of various types). In one embodiment, the transmembranedomain comprises 41BB or CD28, wherein the cytoplasmic domain comprisesan alpha, beta, or zeta chain of the T cell receptor, and wherein theimmune cell is a T cell. In some such embodiments, upon binding thetarget of interest, the T cell is stimulated to initiate intracellularsignaling, produce cytokines, proliferates and degranulates, leading tothe cytotoxic effects on the cancer cell, without the T cells exhibitinga phenotype associated with T cell exhaustion.

Further embodiments provide for a method of treating a subject havingcancer, the method comprising intravenously administering to the subjectan immune cell comprising a chimeric antigen receptor (CAR) expressed ona T cell, wherein the CAR comprises a target binding domain comprising apolypeptide having an amino acid sequence comprising, the polypeptidehaving an amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, or SEQ ID NO:6, however, no cysteine or proline residuesare substituted into any of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, or SEQ ID NO:6, the polypeptide capable of specifically bindinga target of interest expressed by a cancer cell with a binding to thetarget of interest that is greater than binding of a polypeptideaccording to SEQ ID NO:1 to the target of interest, a transmembranedomain selected from 41BB and CD28, and an intracellular domain, whereinthe intracellular domain comprises a signaling domain selected from analpha, beta, or zeta chain of the T cell receptor, wherein, uponadministration to a subject having cancer, the target binding domainspecifically binds to the target of interest expressed by a cancer cell,and wherein the binding of the target of interest induces the T cell togenerate cytotoxic signals that result in cytotoxic effects on thecancer cell. In several embodiments, the cytotoxic effects result fromdegranulation of the T cells. Advantageously, in several embodiments,the activation and cytotoxic activity of the T cells is not associatedwith the T cells exhibiting a phenotype associated with T cellexhaustion. In several embodiments, the CAR optionally further comprisesa second target binding domain comprising a second polypeptide having adifferent target than the target binding domain. In still furtherembodiments, additional targeting domains can optionally be included toenhance binding capacity to a marker, or impart binding specificity toother markers.

Additionally provided for in several embodiments, is the use of animmune cell comprising a chimeric antigen receptor (CAR) for thetreatment of cancer, wherein the CAR comprises a target binding domaincomprising a polypeptide having an amino acid sequence comprising, thepolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, andSEQ ID NO:6, wherein no cysteine or proline residues are substitutedinto any of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQID NO:6, wherein the polypeptide specifically binds a target of interestexpressed by a cancer cell, and wherein the polypeptide's specificbinding to the target of interest is greater than binding of apolypeptide according to SEQ ID NO:1 to the target of interest, atransmembrane domain selected from 41BB and CD28, and an intracellulardomain, wherein the intracellular domain comprises a signaling domainselected from an alpha, beta, or zeta chain of the T cell receptor,wherein, upon administration to a subject having cancer, the targetbinding domain specifically binds to the target of interest expressed bya cancer cell, and wherein the binding of the target of interest inducesthe immune cell to generate cytotoxic signals that result in cytotoxiceffects on the cancer cell. Depending on the embodiment the immune cellscan be a T cell or a natural killer (NK) cell.

In addition to binding domain compositions, methods for generating,screening and using same, there are also provided methods for purifyingtargets of interest. Thus, provided for herein, in several embodiments,is a method for purifying a target of interest comprising contacting asample comprising a target of interest with a composition comprising apolypeptide agent attached to a solid support, wherein the polypeptideagent has an amino acid sequence comprisingMGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN(SEQ ID NO:4), wherein the polypeptide has an amino acid sequence thatdiffers from SEQ ID NO:1, wherein the polypeptide specifically binds thetarget of interest, wherein the polypeptide's specific binding to thetarget of interest is greater than binding of a polypeptide according toSEQ ID NO:1 to the target of interest, the contacting performed underconditions that permit binding of the composition to the target ofinterest, and removing a portion of the sample that is not bound to thecomposition. In several embodiments the method further comprisesdissociating the composition from the target of interest and recoveringthe target of interest. In several embodiments, the target of interestcan be eluted from the composition, thereby purifying (wholly orpartially) the target of interest.

Depending on the embodiment, the solid support may be a bead, a glassslide, a chip, a gelatin, or an agarose. Combinations of supports may beused in certain embodiments. In several embodiments, the polypeptideagent is coupled to the solid support through non-covalent association,while in other embodiments, the polypeptide agent is coupled to thesolid support through covalent bonding. Depending on the embodiment, thesupports, and the target of interest, combinations of covalent andnon-covalent association can also be used.

In several embodiments, the polypeptide agent of the composition furthercomprises a peptide tag, wherein the peptide tag comprises ahexahistidine moiety or a FLAG tag. In some embodiments, the polypeptideagent of the composition further comprises a streptavidin moiety. Othertypes of tags, e.g., enzymes, colorimetric, bioluminescent and/orfluorescent tags can be used, depending on the embodiment.

In some embodiments, the solid support comprises a bead, and thecomposition is suitable for use in affinity chromatography to purify thetarget of interest.

In several embodiments, a nucleic acid molecule encoding the polypeptideis packaged in an expression vector that is used to transduce a cellline to cause the cell line to express the polypeptide. Suchembodiments, allow for production of the polypeptide in larger scale foruse in protein purification.

Also provided for in several embodiments is a method for purifying atarget of interest comprising contacting a sample comprising a target ofinterest with a composition comprising a virus-like particle coupled toa solid support, wherein the virus-like particle expresses a polypeptideas a membrane protein, the polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, and SEQ ID NO:6, wherein the polypeptide has an aminoacid sequence that differs from SEQ ID NO:1, wherein the polypeptidespecifically binds the target of interest, wherein the polypeptide'sspecific binding to the target of interest is greater than binding of apolypeptide according to SEQ ID NO:1 to the target of interest; and thecontacting performed under conditions that permit binding of thecomposition to the target of interest; and removing a portion of thesample that is not bound to the composition. In several embodiments,wherein no cysteine or proline residues are substituted into any of SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 whengenerating the polypeptide.

In several embodiments, the solid support comprises one or more of abead, a glass slide, a chip, a gelatin, or an agarose. In severalembodiments, the polypeptide of the composition further comprises apeptide tag, wherein the peptide tag comprises a hexahistidine moiety ora FLAG tag. As discussed herein, other types of tags may be used inadditional embodiments.

In some embodiments, the portion of the sample that is not bound to thecomposition is discarded. In some embodiments, the portion of the samplethat is not bound to the composition is contacted with the composition asecond time to capture additional target of interest, thereby improvingthe overall yield of the purification.

In several embodiments, the method further comprises contacting theportion of the sample that is not bound to the composition with anantibody directed against the polypeptide of the composition, theantibody being generated from membrane bound virus-like particles (VLP)expressing the polypeptide released from a mammalian cell is engineeredto express a fusion protein comprising the polypeptide fused to achimeric antigen receptor (CAR), the fusion protein being expressed onthe generated VLPs, wherein the antibodies are suitable for use in anassay to detect residual polypeptides detached from the solid support.

Not only are there provided methods for purifying a target (e.g.,removing the target from a larger sample), but several embodimentsprovide for a method for removing one or more contaminants from a samplecomprising a target of interest, the method comprising contacting asample comprising a target of interest with a composition comprising avirus-like particle coupled to a solid support, wherein the virus-likeparticle expresses a polypeptide as a membrane protein, the polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, whereinno cysteine or proline residues are substituted into any of SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, wherein thepolypeptide has an amino acid sequence that differs from SEQ ID NO:1,wherein the polypeptide specifically binds one or more contaminants tobe removed from a sample comprising the target of interest, wherein thepolypeptide's specific binding to one or more contaminants is greaterthan binding of a polypeptide according to SEQ ID NO:1 to the one ormore contaminants; the contacting performed under conditions that permitbinding of the composition to the one or more contaminants; andcollecting a portion of the sample that is not bound to the composition.As discussed above, in several embodiments, the polypeptide of thecomposition further comprises a tag, such as a peptide tag. In severalembodiments, the peptide tag comprises a hexahistindine moiety or a FLAGtag. Depending on the embodiments, the solid support may comprise abead, a glass slide, a chip, a gelatin, or an agarose and the virus-likeparticles are coupled to the solid support through non-covalentassociation. In some embodiments, the portion of the sample thatcollected is contacted with the composition a second time to removeadditional contaminants from the sample.

Also provided for herein are compositions for use in proteinpurification. In several embodiments, there is provided an affinityresin comprising a polypeptide agent having an amino acid sequencecomprising a sequence selected from the group consisting of:MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2),MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN (SEQ ID NO:3),MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN(SEQ ID NO:4),MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN (SEQ ID NO:5),MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN (SEQ ID NO:6),MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7),MGSWAEFKQRLAAIKTRLEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN(SEQ ID NO:8),MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAAFEKEIAAFESELQAYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN(SEQ ID NO:9),MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN (SEQ ID NO:10) andMGSWX₅EFX₈X₉RLX₁₂MX₁₅X₁₆RLX₁₉ALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN (SEQ ID NO:11), as well as combinations thereof,and wherein the amino acid sequence is not SEQ ID NO:1.

In any of the sequences listed above, any of the X positions (e.g.,“X_(n)”) can be a natural or non-natural amino acid; wherein each X_(n)is the same or different natural or non-natural amino acid.Additionally, in several embodiments, Z₁ and/or Z₂ can comprise betweenabout 2 to about 30 natural or non-natural amino acids.

In several embodiments, the polypeptide agent has an amino acid sequencethat differs from SEQ ID NO:1 by an amino acid substitution at one ormore residues. Depending on the embodiments the amino acid substitutionat one or more residues can comprise a conservative substitution, or anon-conservative substitution. Combinations of conservative andnon-conservative substitutions may also be use, in several embodiments.Additionally, in several embodiments, the amino acid substitution at oneor more residues comprises a substitution at a solvent accessibleresidue. In some embodiments, the amino acid substitution at one or moreresidues comprises a substitution at a solvent inaccessible residue. Insome embodiments, substitutions (whether conservative ornon-conservative) can optionally be made at both solvent accessible andsolvent inaccessible residues. In several embodiments, the polypeptideagent has an amino acid sequence that differs from SEQ ID NO:1 by anamino acid deletion at one or more residues.

In several embodiments, there is provided a method of making an affinityresin comprising attaching to a solid support a polypeptide agent havingan amino acid sequence comprising a sequence selected from the groupconsisting of:MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2),MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN (SEQ ID NO:3),MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN(SEQ ID NO:4),MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN (SEQ ID NO:5),MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN (SEQ ID NO:6),MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7),MGSWAEFKQRLAAIKTRLEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN(SEQ ID NO:8),MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAAFEKEIAAFESELQAYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN(SEQ ID NO:9),MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN (SEQ ID NO:10) andMGSWX₅EFX₈X₉RLX₁₂MX₁₅X₁₆RLX₁₉ALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN (SEQ ID NO:11), and combinations thereof, whereinthe amino acid sequence is not SEQ ID NO:1. In several embodiments, theX positions of the sequences (e.g., “X_(n)”) can comprise a natural ornon-natural amino acid; wherein each X_(n) is the same or differentnatural or non-natural amino acid; and/or wherein Z₁ and/or Z₂ is 2 to30 natural or non-natural amino acids. In several embodiments, thepolypeptide agent is attached to the solid support by covalent bonding,by non-covalent association, or combinations thereof. In severalembodiments, the solid support comprises one or more of a bead, glassslide, chip, gelatin, or agarose.

Further provided for protein purification, in several embodiments, is acomposition comprising a solid support coupled to a polypeptide agenthaving an amino acid sequence comprising a sequence selected from thegroup consisting ofMGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2),MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN (SEQ ID NO:3),MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN(SEQ ID NO:4),MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN (SEQ ID NO:5),MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN (SEQ ID NO:6),MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7),MGSWAEFKQRLAAIKTRLEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN(SEQ ID NO:8),MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAAFEKEIAAFESELQAYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN(SEQ ID NO:9),MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN (SEQ ID NO:10) andMGSWX₅EFX₈X₉RLX₁₂MX₁₅X₁₆RLX₁₉ALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN (SEQ ID NO:11), and combinations thereof, whereinthe amino acid sequence is not SEQ ID NO:1. In several embodiments,X_(n) is a natural or non-natural amino acid; wherein each X_(n) is thesame or different natural or non-natural amino acid; and/or Z₁ and/or Z₂is 2 to 30 natural or non-natural amino acids. In several embodiments,the polypeptide agent has an amino acid sequence that differs from SEQID NO:1 by an amino acid substitution at one or more residues.

Depending on the embodiment, the amino acid substitution at one or moreresidues may comprise a conservative substitution or may comprise anon-conservative substitution. Combinations of conservative andnon-conservative substitutions may also be used, in certain embodiments.In several embodiments, the amino acid substitution at one or moreresidues comprises a substitution at a solvent accessible residue. Inseveral embodiments, the amino acid substitution at one or more residuescomprises a substitution at a solvent inaccessible residue. Someembodiments employ substitutions at both solvent accessible andinaccessible residues. In several embodiments, the polypeptide agent hasan amino acid sequence that differs from SEQ ID NO:1 by an amino aciddeletion at one or more residues. Depending on the embodiments, thesolid support may comprise one or more of a bead, glass slide, chip,gelatin, or agarose.

In several embodiments, the polypeptides disclosed herein can be used inprotein analytics, such as function as detectable agents or tags. Assuch, there is provided herein, in several embodiments, a compositioncomprising a polypeptide agent conjugated to a detectable agent and/ortag, wherein the polypeptide agent has an amino acid sequence comprisinga sequence selected from the group consisting of:MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2),MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN (SEQ ID NO:3),MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN(SEQ ID NO:4),MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN (SEQ ID NO:5),MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN (SEQ ID NO:6),MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7),MGSWAEFKQRLAAIKTRLEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN(SEQ ID NO:8),MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAAFEKEIAAFESELQAYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN(SEQ ID NO:9),MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN (SEQ ID NO:10) andMGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN (SEQ ID NO:11), and combinations thereof, whereinthe amino acid sequence is not SEQ ID NO:1. In several embodiments,X_(n) is a natural or non-natural amino acid; wherein each X_(n) is thesame or different natural or non-natural amino acid; and/or wherein Z₁and/or Z2 is 2 to 30 natural or non-natural amino acids.

In several embodiments, the detectable agent comprises a chromogen. Inseveral embodiments, the detectable agent comprises a fluorescent dye.In several embodiments, the detectable agent comprises a radionuclide.In such embodiments, the detectable agent is quantifiable.

In several embodiments of the composition, the polypeptide agent isconjugated to a chromatography bead, resin, glass slide, chip, gelatin,or agarose. In several embodiments, the tag comprises polyhistidyl tag,a myc tag, or a FLAG tag. Combinations of tags may also be used inseveral embodiments. In several embodiments, the polypeptide agent isconjugated to a detectable agent or tag by covalent binding. In severalembodiments of the composition, the polypeptide agent is a fusionprotein. In several embodiments, the polypeptide agent is multimeric.

De novo binding domain (DBD) containing polypeptides (DBDpp) thatspecifically bind targets of interest are provided, as are nucleic acidsencoding the provided DBDpp, vectors containing the nucleic acids andhost cells containing the nucleic acids and vectors. DBDpp libraries,methods for producing and screening such libraries and the DBDppidentified from such libraries and screens are also provided. DBDpp suchas DBDpp fusion proteins, are also provided as are methods of making andusing the DBDpp. Such uses include, but are not limited to, affinitypurification, and diagnostic and therapeutic applications.

In one embodiment, a DBDpp is provided whose amino acid sequence differs(e.g., due to amino acid modifications) from that of a referencescaffold having the sequence of SEQ ID NO: 1. The reference scaffold isa variant of a non-naturally occurring and targetless (e.g., toApplicant's knowledge, no target is presently known) antiparallel threehelical bundle reference polypeptide originally engineered as anexercise in protein folding (see, Walsh et al., PNAS 96:5486-5491 (1999)incorporated by reference herein in its entirety). It has beendiscovered, and is disclosed herein in several embodiments, thatpolypeptides containing modifications of the targetless referencescaffold having the amino acid sequence of SEQ ID NO:1 are able tospecifically bind targets of interest. While not wishing to be bound bytheory, it is believed that in designing the DBD, the structuralconstraints of surface-exposed residues (that can be modified) conferthe ability of the surface exposed residues to specifically bind atarget of interest.

In one embodiment, a DBDpp agent comprises a polypeptide whose aminoacid sequence shows homology with SEQ ID NO:1 but differs from SEQ IDNO:1 by modification of one or more amino acids. According to severalembodiments, the target-binding agents (e.g., the DBDpp) provided hereinspecifically bind to a target of interest (such as a marker associatedwith cancer or a tumor, such as CD123, CD137, PD-L1, CD19, CD22, NY-ESO,MAGE A3, as non-limiting embodiments). In several embodiments, aprovided target-binding agent (e.g., a DBDpp) comprises a total of 5 to25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60amino acid residues that have been modified as compared to SEQ ID NO:1;and wherein the agent specifically binds a target of interest. Inanother embodiment, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to50, 5 to 55, or 5 to 60 of the modified amino acid residues aresubstitutions. In another embodiment, 5 to 25, 5 to 30, 5 to 35, 5 to40, 5 to 45, or 5 to 50 of the modified amino acid residues areconservative substitutions. In another embodiment, 5 to 25, 5 to 30, 5to 35, 5 to 40, 5 to 45, or 5 to 50 of the modified amino acid residuesare non-conservative substitutions. In a further embodiment, 5 to 15, 5to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of the amino acidresidue modifications are conservative substitutions and 5 to 15, 5 to20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of the amino acidresidue modifications are non-conservative substitutions. In additionalembodiments, 1 to 25, 1 to 30, 1 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to55, or 5 to 60 of the substitutions are at amino acid residues of SEQ IDNO:1 selected from the group consisting of: M1, G2, S3, W4, A5, E6, K8,Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23, S24, E25,A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44,Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61,A62, R64, D65, E66, Q68, A69, Y70, R71, H72, and N73. In a furtherembodiment, 1 to 20, 1 to 30, or 1 to 40 of the substitutions are atamino acid residues of SEQ ID NO:1 selected from the group consistingof: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20,A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54,A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, and Y70. In anoptional further embodiment, the DBDpp optionally further comprises anamino acid sequence wherein 1 to 5, 1 to 10, 1 to 15, 5 to 10 or 5 to 15of the residues corresponding to the solvent inaccessible residues ofthe amino acid sequence of SEQ ID NO:1 are substituted and wherein theDBDpp specifically binds a target of interest. In several embodiments,the DBDpp comprise an amino acid sequence wherein about 1 to about 5,about 1 to about 10, about 1 to about 15, about 5 to about 10, about 5to about 15 (or more) of the residues that correspond to the solventaccessible or the solvent inaccessible residues of the amino acidsequence of SEQ ID NO:1 are substituted. In several embodiments, thesubstitution of both accessible and inaccessible residues confers agreater degree of target specificity as compared to substitution of onlyaccessible or only inaccessible residues. In a further optionalembodiment, the substituted residues corresponding to a solventinaccessible residue of SEQ ID NO:1 are selected from the groupconsisting of: F7, L11, I14, L18, L28, F31, I35, F38, L42, V53, L56,A60, I63, and L67, and Y70. In an additional embodiment, L21 and Y45 arealso included in the group of substituted, solvent inaccessibleresidues. In an additional embodiment, the DBDpp is a fusion protein(e.g., the DBDpp is fused, conjugated, or otherwise associated withanother molecule, directly or indirectly, such as a therapeutic ordiagnostic agent). In one embodiment, the DBDpp is attached to a solidsupport. In a further embodiment, the solid support is selected from thegroup consisting of: a bead, a glass slide, a chip, a gelatin, and anagarose. In an additional embodiment, the DBDpp specifically binds atarget of interest selected from the group consisting of: a nucleicacid, an oligosaccharide, a peptide, a protein, a cell surface antigen,and a small organic molecule. In a further embodiment, the DBDppspecifically binds a protein selected from the group consisting of: animmunoglobulin, an enzyme, a hormone, a serum protein, a cell surfaceprotein, a therapeutic protein, a tumor-specific antigen (TSA), acancer-specific antigen (CSA), and a protein containing a peptide tag.In another embodiment, the DBDpp specifically binds a target disclosedherein. Nucleic acids encoding the DBDpp and vectors containing thenucleic acids are also provided. Host cells (including viral particles)containing the nucleic acids and vectors are also provided. In someembodiments, the host cell displays the DBDpp on its surface. Inadditional embodiments, the host cell is a prokaryote or a eukaryotethat display the DBDpp on its surface. In a further embodiment, the hostcell is a phage that displays the DBDpp on its surface. In a furtherembodiment, the host cell is a human immune cell that expresses a DBDppfusion protein on its surface. Libraries comprising a plurality of DBDppare also provided.

In one embodiment, a DBDpp comprises an amino acid sequence selectedfrom the group consisting of: (a)MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN (SEQ ID NO:4), whereinX₅, X₈, X₉, X₁₂, X₁₅, X₁₆, X₁₉, X₅₅, X₅₈, X₅₉, X₆₂, X₆₅, and/or X₆₆, isa natural and/or non-natural amino acid residue; (b)MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALRKEAAAIRDELQAYRHN(SEQ ID NO:2), wherein X₅, X₆, X₉, X₁₀, X₁₃, X₁₆, X₁₇, X₃₀, X₃₃, X₃₄,X₃₇, X₄₀, X₄₁, and/or X₄₄, is a natural and/or non-natural amino acidresidue; (c) MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN (SEQID NO:3), wherein X₃₂, X₃₃, X₃₆, X₃₉, X₄₀, X₄₃, X₅₇, X₅₈, X₆₁, X₆₄, X₆₅,and/or X₆₈, is a natural and/or non-natural amino acid residue, and; (d)MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN (SEQ ID NO:5), wherein X₅, X₆, X₉, X₁₀, X₁₃, X₁₆,X₁₇, X₃₂, X₃₃, X₃₆, X₃₉, X₄₀, X₄₃, X₅₅, X₅₈, X₅₉, X₆₂, X₆₅, and/or X₆₆,is a natural and/or non-natural amino acid residue; and (e)MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN(SEQ ID NO:6), wherein X₅, X₈, X₉, X₁₂, X₁₅, X₁₆, X₁₉, X₃₀, X₃₃, X₃₄,X₃₇, X₄₀, X₄₁, X₄₄, X₅₇, X₅₈, X₆₁, X₆₄, X₆₅, and/or X₆₈, is a naturaland/or non-natural amino acid residue; and wherein the DBDppspecifically binds a target of interest. In several embodiments, a DBDppcomprises, consists of, or consists essentially of an amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, and SEQ ID NO:5. In an additional embodiment, X_(n) is anatural amino acid residue. In a further embodiment, X_(n) is a naturalamino acid residue other than cysteine or proline. In still additionalembodiments, X_(n) is a deletion of an amino acid (e.g., optionally anull position in the sequence). In an additional embodiment, the DBDppis a fusion protein. In another embodiment, the DBDpp specifically bindsa target of interest selected from the group consisting of: a nucleicacid, an oligosaccharide, a peptide, a protein, a cell surface antigen,and a small organic molecule. In a further embodiment, the DBDppspecifically binds a protein selected from the group consisting of: animmunoglobulin, an enzyme, a hormone, a serum protein, a cell surfaceprotein, a therapeutic protein, a TSA, a CSA, and a protein containing apeptide tag. In a further embodiment, the DBDpp specifically binds atarget disclosed herein. In an additional embodiment, a librarycontaining a plurality of DBDpp is provided. Nucleic acids encoding theDBDpp and vectors containing the nucleic acids are also provided. Hostcells (including viral particles) containing the nucleic acids andvectors are also provided. In some embodiments, the host cell is aprokaryote or a eukaryote that display the DBDpp on its surface. In someembodiments, the host cell displays the DBDpp on its surface. In afurther embodiment, the host cell is a phage that displays the DBDpp onits surface. In a further embodiment, the host cell is a human immunecell (e.g., B-cell, T-cell, killer T-cell, helper T-cell, regulatoryT-cell, antigen presenting cell, natural killer cell, and the like) thatexpresses one or more DBDpp fusion proteins on its surface. In oneembodiment, the DBDpp is attached to a solid support. In a furtherembodiment, the solid support is selected from the group consisting of:a bead, a glass slide, other glass or plastic-based materials (e.g., afilter or filter device), a filtration material (e.g., glass fiber,steel wool, polyethersulfone, etc.), a chip, a gelatin, and an agarose,and combinations thereof.

Also provided is an isolated DBDpp that comprises an amino acid sequenceselected from the group consisting of: (a)MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAAFEKEIAAFESELQAYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN (SEQ ID NO:9),wherein X₅, X₈, X₉, X₁₂, X₁₅, X₁₆, X₁₉, X₅₀, X₅₃, X₅₄, X₅₇, X₆₀, and/orX₆₁, is a natural and/or non-natural amino acid residue, and Z₁ and/orZ₂ is 2 to 30 natural and/or non-natural amino acid residues; (b)MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALRKEAAAIRDELQAYRHN(SEQ ID NO:7), wherein X₅, X₆, X₉, X₁₀, X₁₃, X₁₆, X₁₇, X₂₈, X₃₁, X₃₂,X₃₅, X₃₈, X₃₉, and/or X₄₂, is a natural and/or non-natural amino acidresidue, and Z₁ and/or Z₂ is 2 to 30 natural and/or non-natural aminoacid residues; (c)MGSWAEFKQRLAAIKTRLEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN(SEQ ID NO:8), wherein X₃₀, X₃₁, X₃₄, X₃₇, X₃₈, X₄₁, X₅₂, X₅₃, X₅₆, X₅₉,X₆₀, and/or X₆₃, is a natural and/or non-natural amino acid residue, andZ₁ and/or Z₂ is 2 to 30 natural and/or non-natural amino acid residues;(d)MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN(SEQ ID NO:10), wherein X₅, X₆, X₉, X₁₀, X₁₃, X₁₆, X₁₇, X₃₀, X₃₁, X₃₄,X₃₇, X₃₈, X₄₁, X₅₀, X₅₃, X₅₄, X₅₇, X₆₀, and/or X₆₁, is a natural and/ornon-natural amino acid residue, and Z₁ and/or Z₂ is 2 to 30 naturaland/or non-natural amino acid residues; and (e)MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN (SEQ ID NO:11), wherein X₅, X₈, X₉, X₁₂,X₁₅, X₁₆, X₁₉, X₂₈, X₃₁, X₃₂, X₃₅, X₃₈, X₃₉, X₄₂, X₅₂, X₅₃, X₅₆, X₅₉,X₆₀, and/or X₆₃, is a natural and/or non-natural amino acid residue, andZ₁ and/or Z₂ is 2 to 30 natural and/or non-natural amino acid residues;and wherein the DBDpp specifically binds a target of interest. Inseveral embodiments, a DBDpp comprises, consists of, or consistsessentially of an amino acid sequence selected from the group consistingof SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, and SEQ IDNO:11. In an additional embodiment, X_(n) is a natural amino acidresidue. In a further embodiment, X_(n) is a natural amino acid residueother than cysteine or proline. In still additional embodiments, X_(n)is a deletion of an amino acid (e.g., optionally a null position in thesequence). In still additional embodiments, Z₁ and/or Z₂ are deletionsof amino acids (e.g., optionally null positions in the sequence). In anadditional embodiment, the DBDpp is a fusion protein. In anotherembodiment, the DBDpp specifically binds a target of interest selectedfrom the group consisting of: a nucleic acid, an oligosaccharide, apeptide, a protein, a cell surface antigen, and a small organicmolecule. In a further embodiment, the DBDpp specifically binds aprotein selected from the group consisting of: an immunoglobulin, anenzyme, a hormone, a serum protein, a cell surface protein, atherapeutic protein, a TSA, a CSA, and a protein containing a peptidetag. In a further embodiment, the DBDpp specifically binds a targetdisclosed herein. In an additional embodiment, a library containing aplurality of DBDpp is provided. Nucleic acids encoding the DBDpp andvectors containing the nucleic acids are also provided. Host cells,including viral particles, containing the nucleic acids are alsoprovided. In some embodiments, the host cell displays the DBDpp on itssurface. In a further embodiment, the host cell is a phage that displaysthe DBDpp on its surface. In additional embodiments, the host cell is aprokaryote or a eukaryote that display the DBDpp on its surface. In afurther embodiment, the host cell is a human immune cell that expressesa DBDpp fusion protein on its surface. In one embodiment, the DBDpp isattached to a solid support. In a further embodiment, the solid supportis selected from the group consisting of: a bead, a glass slide, a chip,a gelatin, and an agarose.

Nucleic acids encoding a DBDpp such as a DBDpp fusion protein are alsoprovided. Additionally provided are vectors containing nucleic acidsencoding DBDpp (e.g., DBDpp fusion proteins) and host cells containingthe nucleic acids and vectors. In some embodiments, the host cell is aviral particle, or a bacterial, yeast, fungal, or plant cell. In aparticular embodiment, the host cell is a mammalian cell. In anotherembodiment, the mammalian cell is an immune cell. In a furtherembodiment, the host cell is a human immune cell. In some embodiments,the host cell displays the DBDpp as a fusion protein on the cellsurface. In a further embodiment, the host cell is a human immune cellthat displays a DBDpp on the cell surface. Additionally provided hereinare vector libraries comprising nucleic acids encoding a plurality ofDBDpp.

Also provided is a library containing a plurality of DBDpp. In oneembodiment, the DBDpp library comprises a plurality of DBDpp containinga different amino acid sequences and that comprise the amino acidsequence of SEQ ID NO:1 wherein a total of 5 to 25, 5 to 30, 5 to 35, 5to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 amino acid residues(including any number between those listed) have been modified; andwherein the DBDpp specifically binds a target of interest. In anotherembodiment, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to55, or 5 to 60 (including any number between those listed) of themodified amino acid residues are substitutions. In another embodiment, 5to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, or 5 to 50 (including anynumber between those listed) of the modified amino acid residues areconservative substitutions. In another embodiment, 5 to 25, 5 to 30, 5to 35, 5 to 40, 5 to 45, or 5 to 50 (including any number between thoselisted) of the modified amino acid residues are non-conservativesubstitutions. In a further embodiment, 5 to 15, 5 to 20, 5 to 25, 5 to30, 5 to 35, 5 to 40, or 5 to 45 (including any number between thoselisted) of the amino acid residue modifications are conservativesubstitutions and 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40,or 5 to 45 (including any number between those listed) of the amino acidresidue modifications are non-conservative substitutions. In additionalembodiments, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to55, or 5 to 60 (including any number between those listed) of thesubstitutions are at one or more amino acid residues of SEQ ID NO:1selected from the group consisting of: Ml, G2, S3, W4, A5, E6, K8, Q9,R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23, S24, E25, A26,E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45,K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61, A62,R64, D65, E66, Q68, A69, Y70, R71, H72, and N73. In a furtherembodiment, 1 to 20, 1 to 30, or 1 to 40 (including any number betweenthose listed) of the substitutions are at one or more of amino acidresidues of SEQ ID NO:1 selected from the group consisting of: G2, S3,W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30,E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57,K58, E59, A61, A62, R64, D65, E66, Q68, A69, and Y70. In anotherembodiment, the library comprises at least 2, 3, 4, 5, 10, 25, 50, 75,100, 250, 500, or 1000 (including any range between those numberslisted, such as 2-10, 5-25, 50-100, 250-1000, etc.) different DBDpp thatspecifically binding different targets (or DBDpp that have differentialspecificity for a given target). In a further embodiment, the differenttargets bound by DBDpp in the library are selected from the groupconsisting of: a nucleic acid, an oligosaccharide, a peptide, a protein,a cell surface antigen, and a small organic molecule. In a furtherembodiment, the library comprises at least 2, 3, 4, 5, 10, 25, 50, 75,100, 250, 500, or 1000 (including any range between those numberslisted, such as 2-10, 5-25, 50-100, 250-1000, etc.) different DBDpp thatspecifically bind a protein target selected from the group consistingof: an immunoglobulin, an enzyme, a hormone, a serum protein, a cellsurface protein, a therapeutic protein, a TSA, a CSA, and a proteincontaining a peptide tag. In a further embodiment, the library comprisesat least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 (includingany range between those numbers listed, such as 2-10, 5-25, 50-100,250-1000, etc.) different DBDpp that specifically bind a targetdisclosed herein. In an additional embodiment, the library is a vectorlibrary or a host cell library. In an additional embodiment, the vectorlibrary is a library of host cells. In another embodiment, the host celllibrary comprises a plurality of host cells that display the DBDpp ontheir surface. In a further embodiment, the host cells are phage thatdisplay the DBDpp on their surface. In some embodiments, the vectorlibrary comprises: (a) nucleic acids encoding 3 DBDpp that specificallybind to different targets; (b) nucleic acids encoding 3 DBDpp havingdifferent sequences that specifically bind to the same target; (c)nucleic acids encoding 3 DBDpp having different sequences thatspecifically bind to the same epitope of a target; (d) nucleic acidsencoding 3 DBDpp having different sequences that specifically bind todifferent epitopes of a target; (e) nucleic acids encoding 3 DBDpphaving different sequences that compete for binding to the same target;or (f) 3 different nucleic acid sequences encoding the same DBDppsequence. Host cells containing the vectors are also provided.

Also provided is a vector library comprising a plurality of differentnucleic acid sequences encoding DBDpp, that comprise the amino acidsequence of SEQ ID NO:1 wherein a total of 1 to 5, 5 to 25, 5 to 30, 5to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 amino acidresidues have been modified (or any number in between those listed); andwherein the DBDpp specifically binds a target of interest. In anotherembodiment, 1 to 5, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to50, 5 to 55, or 5 to 60 of the modified amino acid residues (or anynumber in between those listed) encoded by the nucleic acids sequencesare substitutions. In another embodiment, 1 to 5, 5 to 25, 5 to 30, 5 to35, 5 to 40, 5 to 45, or 5 to 50 of the modified amino acid residues (orany number in between those listed) are conservative substitutions. Inanother embodiment, 1 to 5, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45,or 5 to 50 of the encoded modified amino acid residues (or any number inbetween those listed) are non-conservative substitutions. In a furtherembodiment, 1 to 5, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to40, or 5 to 45 of the encoded amino acid residue modifications (or anynumber in between those listed) are conservative substitutions and 1 to5, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 ofthe encoded amino acid residue modifications (or any number in betweenthose listed) are non-conservative substitutions. In additionalembodiments, 1 to 5, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to50, 5 to 55, or 5 to 60 of the encoded substitutions (or any number inbetween those listed) are at amino acid residues of SEQ ID NO:1 selectedfrom the group consisting of one or more of: Ml, G2, S3, W4, A5, E6, K8,Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23, S24, E25,A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44,Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58, E59, A61,A62, R64, D65, E66, Q68, A69, Y70, R71, H72, and N73. In a furtherembodiment, 1 to 20, 1 to 30, or 1 to 40 of the encoded substitutions(or any number in between those listed) are at amino acid residues ofSEQ ID NO:1 selected from the group consisting of one or more of: G2,S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29,A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55,R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, and Y70. In a furtherembodiment, the nucleic acids optionally encode a DBDpp that furthercomprises an amino acid sequence wherein 1 to 5, 5 to 15, 5 to 20, 5 to25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 (or any number in betweenthose listed) of the residues corresponding to the solvent inaccessibleresidues of the amino acid sequence of SEQ ID NO:1 are substituted andwherein the DBDpp specifically binds a target of interest. In anotherembodiment, the library comprises nucleic acids encoding at least 2, 3,4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp thatspecifically bind different targets (or have varied affinity for thesame target). In a further embodiment, the different targets bound byDBDpp in the library are selected from the group consisting of: anucleic acid, an oligosaccharide, a peptide, a protein, a cell surfaceantigen, and a small organic molecule. In a further embodiment, thelibrary comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25,50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind aprotein target selected from the group consisting of: an immunoglobulin,an enzyme, a hormone, a serum protein, a cell surface protein, atherapeutic protein, a TSA, a CSA, and a protein containing a peptidetag. In a further embodiment, the library comprises nucleic acidsencoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000different DBDpp that specifically bind a target disclosed herein. In anadditional embodiment, the vector library is contained in host cells(e.g., viral particles). In another embodiment, the library comprises aplurality of host cells that display the DBDpp on their surface. In afurther embodiment, the host cells are phage that display the DBDpp ontheir surface. In some embodiments, the vector library comprises: (a)nucleic acids encoding 3 DBDpp that specifically bind to differenttargets; (b) nucleic acids encoding 3 DBDpp having different sequencesthat specifically bind to the same target; (c) nucleic acids encoding 3DBDpp having different sequences that specifically bind to the sameepitope of a target; (d) nucleic acids encoding 3 DBDpp having differentsequences that specifically bind to different epitopes of a target; (e)nucleic acids encoding 3 DBDpp having different sequences that competefor binding to the same target; or (f) 3 different nucleic acidsequences encoding the same DBDpp sequence. Host cells containing thevectors are also provided.

In one embodiment, a vector library comprises a plurality of differentnucleic acids encoding DBDpp, wherein the encoded DBDpp comprises anamino acid sequence selected from the group consisting of: (a)MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN (SEQ ID NO:4),wherein X₅, X₈, X₉, X₁₂, X₁₅, X₁₆, X₁₉, X₅₅, X₅₈, X₅₉, X₆₂, X₆₅, and/orX₆₆, is a natural and/or non-natural amino acid residue; (b)MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ IDNO:2), wherein X₅, X₆, X₉, X₁₀, X₁₃, X₁₆, X₁₇, X₃₀, X₃₃, X₃₄, X₃₇, X₄₀,X₄₁, and/or X₄₄, is a natural and/or non-natural amino acid residue; (c)MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN(SEQ ID NO:3), wherein X₃₂, X₃₃, X₃₆, X₃₉, X₄₀, X₄₃, X₅₇, X₅₈, X₆₁, X₆₄,X₆₅, and/or X₆₈, is a natural and/or non-natural amino acid residue,and; (d)MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN (SEQ ID NO:5), wherein X₅, X₆, X₉, X₁₀,X₁₃, X₁₆, X₁₇, X₃₂, X₃₃, X₃₆, X₃₉, X₄₀, X₄₃, X₅₅, X₅₈, X₅₉, X₆₂, X₆₅,and/or X₆₆, is a natural and/or non-natural amino acid residue; and (e)MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN(SEQ ID NO:6), wherein X₅, X₈, X₉, X₁₂, X₁₅, X₁₆, X₁₉, X₃₀, X₃₃, X₃₄,X₃₇, X₄₀, X₄₁, X₄₄, X₅₇, X₅₈, X₆₁, X₆₄, X₆₅, and/or X₆₈, is a naturaland/or non-natural amino acid residue; and wherein the DBDppspecifically binds a target of interest. In an additional embodiment,X_(n) is a natural amino acid residue. In a further embodiment, X_(n) isa natural amino acid residue other than cysteine or proline. In stilladditional embodiments, X_(n) is a deletion of an amino acid (e.g.,optionally a null position in the sequence). In an additionalembodiment, a plurality of the vectors in the library encode a DBDppfusion protein. In another embodiment, the library comprises nucleicacids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or1000 different DBDpp that specifically bind different targets. In afurther embodiment, the different targets bound by DBDpp encoded by thenucleic acids in the library are selected from the group consisting of:a nucleic acid, an oligosaccharide, a peptide, a protein, a cell surfaceantigen, and a small organic molecule. In a further embodiment, thelibrary comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25,50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind aprotein target selected from the group consisting of: an immunoglobulin,an enzyme, a hormone, a serum protein, a cell surface protein, atherapeutic protein, a TSA, a CSA, and a protein containing a peptidetag. In a further embodiment, the library comprises nucleic acidsencoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000different DBDpp that specifically bind a target disclosed herein. In anadditional embodiment, a plurality of the vectors of the vector libraryare contained in host cells (e.g., viral particles such as phage), E.coli, yeast, and mammalian cells. In another embodiment, the host cellsdisplay DBDpp on their surface. In a further embodiment, the host cellsare phage that display DBDpp on their surface. In some embodiments, thevector library comprises: (a) nucleic acids encoding 3 DBDpp thatspecifically bind to different targets; (b) nucleic acids encoding 3DBDpp having different sequences that specifically bind to the sametarget; (c) nucleic acids encoding 3 DBDpp having different sequencesthat specifically bind to the same epitope of a target; (d) nucleicacids encoding 3 DBDpp having different sequences that specifically bindto different epitopes of a target; (e) nucleic acids encoding 3 DBDpphaving different sequences that compete for binding to the same target;or (f) 3 different nucleic acid sequences encoding the same DBDppsequence. Host cells containing the vectors are also provided.

In one embodiment, a vector library comprises a plurality of nucleicacids encoding DBDpp comprising an amino acid sequence selected from thegroup consisting of: (a)MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAAFEKEIAAFESELQAYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN (SEQ ID NO:9), wherein X₅, X₈, X₉, X₁₂,X₁₅, X₁₆, X₁₉, X₅₀, X₅₃, X₅₄, X₅₇, X₆₀, and/or X₆₁, is a natural and/ornon-natural amino acid residue, and Z₁ and Z₂ is 2 to 30 natural and/ornon-natural amino acid residues; (b) MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7), wherein X₅, X₆, X₉, X₁₀, X₁₃, X₁₆, X₁₇, X₂₈,X₃₁, X₃₂, X₃₅, X₃₈, X₃₉, and/or X₄₂, is a natural and/or non-naturalamino acid residue, and Z₁ and Z₂ is 2 to 30 natural and/or non-naturalamino acid residues; (c) MGSWAEFKQRLAAIKTRLEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN (SEQID NO:8), wherein X₃₀, X₃₁, X₃₄, X₃₇, X₃₈, X₄₁, X₅₂, X₅₃, X₅₆, X₅₉, X₆₀,and/or X₆₃, is a natural and/or non-natural amino acid residue, and Z₁and Z₂ is 2 to 30 natural and/or non-natural amino acid residues; (d)MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN(SEQ ID NO:10), wherein X₅, X₆, X₉, X₁₀, X₁₃, X₁₆, X₁₇, X₃₀, X₃₁, X₃₄,X₃₇, X₃₈, X₄₁, X₅₀, X₅₃, X₅₄, X₅₇, X₆₀, and/or X₆₁, is a natural and/ornon-natural amino acid residue, and Z₁ and Z₂ is 2 to 30 natural and/ornon-natural amino acid residues; and (e) MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN(SEQ ID NO:11), wherein X₅, X₈, X₉, X₁₂, X₁₅, X₁₆, X₁₉, X₂₈, X₃₁, X₃₂,X₃₅, X₃₈, X₃₉, X₄₂, X₅₂, X₅₃, X₅₆, X₅₉, X₆₀, and/or X₆₃, is a naturaland/or non-natural amino acid residue, and Z₁ and Z₂ is 2 to 30 naturaland/or non-natural amino acid residues; and wherein the DBDppspecifically binds a target of interest. In an additional embodiment,X_(n) is a natural amino acid residue. In a further embodiment, X_(n) isa natural amino acid residue other than cysteine or proline. In stilladditional embodiments, X_(n) is a deletion of an amino acid (e.g.,optionally a null position in the sequence). In an additionalembodiment, a plurality of the vectors in the library encode a DBDppfusion protein. In another embodiment, the library comprises nucleicacids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or1000 different DBDpp that specifically bind different targets. In afurther embodiment, the different targets bound by DBDpp encoded by thenucleic acids in the library are selected from the group consisting of:a nucleic acid, an oligosaccharide, a peptide, a protein, a cell surfaceantigen, and a small organic molecule. In a further embodiment, thelibrary comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25,50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind aprotein target selected from the group consisting of: an immunoglobulin,an enzyme, a hormone, a serum protein, a cell surface protein, atherapeutic protein, a TSA, a CSA, and a protein containing a peptidetag. In a further embodiment, the library comprises nucleic acidsencoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000different DBDpp that specifically bind a target disclosed herein. In anadditional embodiment, a plurality of the vectors of the vector libraryare contained in host cells. [In another embodiment, the host cells(e.g., viral particles) display DBDpp on their surface. In a furtherembodiment, the host cells are phage that display DBDpp on theirsurface. In some embodiments, the host cells are mammalian cells. Insome embodiments, the vector library comprises: (a) nucleic acidsencoding 3 DBDpp that specifically bind to different targets; (b)nucleic acids encoding 3 DBDpp having different sequences thatspecifically bind to the same target; (c) nucleic acids encoding 3 DBDpphaving different sequences that specifically bind to the same epitope ofa target; (d) nucleic acids encoding 3 DBDpp having different sequencesthat specifically bind to different epitopes of a target; (e) nucleicacids encoding 3 DBDpp having different sequences that compete forbinding to the same target; or (f) 3 different nucleic acid sequencesencoding the same DBDpp sequence. Host cells containing the vectors arealso provided.

The DBDpp according to several embodiments provided herein possessactivities that include but are not limited to target binding, theability to bind, link, and/or otherwise associate with a target ofinterest (e.g., a purification target, a therapeutic target, adiagnostic target, a peptide tag, and a serum protein such as humanserum albumin (HSA) or an immunoglobulin) in vitro or in vivo and theability to serve as a reactive site for linking or associating proteinssuch as DBDpp fusion proteins with additional moieties (e.g., a solidsupport), and/or other modifications. The DBDpp provided herein can alsopossess additional desirable properties and/or functionalities useful inmanufacturing, purification, formulation and biological, diagnostic, andtherapeutic applications.

In some embodiments, a DBDpp is used to bind, detect, quantitate,remove, and/or purify a target of interest in a sample containing thetarget.

One non-limiting embodiment provides a method for detecting a target ofinterest in a sample, comprising: (a) contacting the sample with a DBDppthat specifically binds the target, under conditions suitable forspecific binding of the DBDpp to the target, to form a target/DBDppcomplex, and (b) detecting the presence of the complex and/or capturedtarget. In one embodiment, the DBDpp is immobilized on a solid support.

Also provided is a method for quantifying a target of interest in asample containing the target, comprising: (a) contacting the sample witha DBDpp that specifically binds the target and that is immobilized on asolid support, under conditions suitable for specific binding of theDBDpp to the target, to form a target/DBDpp complex and (b) detectingthe presence of the target/DBDpp complex and/or captured target, whereinquantitative detection of the product indicates, or is otherwise able tobe correlated with, the quantity of the target in the sample.

Some embodiments provide methods for purifying a target of interest froma sample containing the target that comprises: (a) contacting a samplecontaining a target of interest with a DBDpp that specifically binds thetarget, under conditions suitable for specific binding of the DBDpp tothe target, and (b) recovering the bound target. In some embodiments,the target is recovered by elution. In one embodiment, the DBDpp isimmobilized on a solid support. In a further embodiment, the elution ofthe bound target is monitored by ultra violet light absorption, or othervisualization or chemical-based detection technique. In someembodiments, methods are provided to remove an undesired target ofinterest from a sample and wherein the bound undesired target isdiscarded directly or is eluted (or otherwise collected or separated)and then discarded.

An additional embodiment provides a method of screening a library ofDBDpp for a DBDpp that specifically binds a target of interest, thatcomprises: (a) obtaining a plurality of host cells (e.g., viralparticles, phage, bacteria, and/or mammalian cells) displaying a libraryof DBDpp on their surface; (b) contacting the plurality of host cellswith a target of interest under conditions suitable for specific bindingof the target to a DBDpp; and (c) determining the binding of the targetto the DBDpp. In one embodiment, the host cells are phage that displaythe DBDpp on their surface.

Methods of using DBDpp in diagnostic and therapeutic applications arealso provided, in several embodiments. One embodiment provides a methodof treating a disease or disorder comprising administering atherapeutically effective amount of a DBDpp (e.g., a DBDpp fusionprotein) that specifically binds a therapeutic target of interest to asubject in need thereof. In some embodiments, the disease or disorder iscancer, a disease or disorder of the immune system, or an infection.Methods of treating a disease or disorder that comprisesco-administering an additional therapeutic agent along with a DBDpp arealso provided.

Additionally provided are methods for treating or preventing cancercomprising administering a DBDpp-CAR T lymphocyte to a patient (e.g.,predisposed to or having a cancer) that expresses a tumor antigen on thesurface of target cells, and wherein the DBDpp specifically binds theantigen.

Certain methods summarized above and set forth in further detail belowdescribe certain actions taken by a practitioner; however, it should beunderstood that they can also include the instruction of those actionsby another party. Thus, actions such as “administering a T cellcomprising a target specific binding polypeptide-CAR” include“instructing the administration of a T cell comprising a target specificbinding polypeptide-CAR.”

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B. Schematic depicting SEQ ID NO:1 derived homology model ofDBDpp. Transverse view illustrating the helices and three faces ofdomain (FIG. 1A). Longitudinal view illustrating position of residueE19, N-terminus (NT) and C-terminus (CT) (FIG. 1B).

FIGS. 2A-J. Schematic representation of different homology models ofDBDpp based off the reference scaffold of SEQ ID NO:1. The residuestargeted for modification in the Face libraries (F1, F2, and F3) andCombined libraries (C1 and C2) of DBDpp are darkly shaded. Longitudinaland transverse perspective views of the F1 library are shown in FIG. 2Aand FIG. 2B respectively. Longitudinal and transverse perspective viewsof the F2 library are shown in FIG. 2C and FIG. 2D respectively.Longitudinal and transverse perspective views of the F3 library areshown in FIG. 2E and FIG. 2F respectively. Longitudinal and transverseperspective views of the C1 library are shown in FIG. 2G and FIG. 2Hrespectively. Longitudinal and transverse perspective views of the C2library are shown in FIG. 2I and FIG. 2J respectively. N-terminus (NT)and C-terminus (CT) for each model are indicated.

FIGS. 3A-D. FIG. 3A. Schematic depiction of phage display construct foruse in accordance with several embodiments disclosed herein. FIG. 3B.Depicts a linear vector map for the pComb phagemid vector used togenerate the libraries disclosed herein. The libraries were createdthrough Kunkel mutagenesis, utilizing oligos containing NNK or trimercodons. DBDpp variant peptide sequences were expressed in-frame, betweenthe FLAG peptide tag sequence and M13 gene pIII. The DBDpp wereexpressed as an N-terminal pIII gene fusion, under the control of a DsbAsignal peptide. FIG. 3C. Depicts a linear vector map for the pCombphagemid vector used to generate DBDpp libraries described in theexamples. DBDpp were expressed in-frame, between the DsbA signal peptideand M13 gene pIII. The modified pComb phagemid vector is the same asthat depicted in FIG. 3B, but absent the FLAG peptide tag sequence,which is in accordance with certain embodiments disclosed herein(wherein the FLAG tag is optionally removed or replaced with anothervariety of tag). FIG. 3D depicts data from a comparative binding assay.N-terminal FLAG tag fusions were expressed and purified from E. colicultures. ELISA-based binding assessment demonstrated that purifiedFLAG-pb04 (targets PD-L1) binds in a dose dependent manner to PD-L1-Fccoated microtiter wells, whereas FLAG-α3D (the reference sequence of SEQID 49, with an N-terminal FLAG tag) exhibits no change in binding.

FIGS. 4A-D. DBDpp have novel binding specificities and impart thesenovel binding specificities to another molecule (e.g., an antibody) aspart of a fusion protein (e.g., an antibody-DBDpp fusion protein).Schematic depicting the recombinant fusion of DBDpp (shown as circle) tothe C-terminus (FIG. 4A) and N-terminus (FIG. 4B) of an antibody heavychain. DBDpp—antibody fusions were created using an RSV-specificantibody (SYN) and either the targetless peptide of SEQ ID NO:1 (DBD) orthe CD137-specific DBDpp (bb10). The DBDpp are fused to the N-terminus(bb10-SYN and DBD-SYN) or the C-terminus (SYN-bb10 and SYN-DBD). Allfour antibody fusions bind to RSV (FIG. 4C). However, the fusion of bb10to either the N-terminus (bb10-SYN) or C-terminus (SYN-bb10) of theantibody heavy chain imparts a novel CD137 binding specificity to anotherwise mono-specific antibody (FIG. 4D).

FIGS. 5A-5C. FIG. 5A. Depicts a schematic representation of DBDpp-CARfusion proteins according to several embodiments disclosed herein. Sixdifferent DBDpp-CAR formats are presented, by way of example, and areintended to be illustrative and not limiting. Extracellular DBDppdomains may be specific for a single target (e.g. DBDpp “A”) or morethan one target or epitope (e.g. DBDpp “A” and DBDpp “B”). Non-limitingexamples of transmembrane (TM) domains are shown, as are non-limitingexamples of intracellular domains derived from CD3, CD28 and 41BB.Domains are optionally linked via peptide linkers (shown in shading).FIG. 5B. Depicts a further schematic of a membrane bound (e.g.,extracellular) DBDpp-CAR fusion. FIG. 5C. Depicts a schematic of asoluble DBDpp.

FIGS. 6A-C. Multi-specific DBDpp fusions recognize cell surface targets.FACS analysis indicates that bb10-SYN and SYNbb10 bispecific antibodiesbind (shaded histogram) to activated CEM cell at levels greater than SYNalone (black outline). The weaker binding observed with the bb10N-terminal fusion (FIG. 6A) as compared to the C-terminal fusion (FIG.6B) is consistent with the above ELISA data. URE1 is a recombinantantibody constructed formed from variable domains of theCD137-targeting, urelumab fused to IgG scaffold. Binding of CEM cellswas performed after activated with PMA (50 ng/ml) and ionomycin (500ng/ml) for 48 hr. Detection of bound antibody was performed withanti-IgG1 Fc (FITC-A).

FIGS. 7A-D. DBDpp impart novel biological activity to an antibody-DBDppfusion protein. Activation of CD137 by ligand or agonistic antibodies,such as urelumab, induces a signaling cascade that results in cytokineproduction, expression of anti-apoptotic molecules, and enhanced immuneresponses. The agonistic potential of the CD137-targeting DBDpp, bb10was assessed by measuring the ability of bb10-SYN and SYN-bb10 to inducecytokine release from PBMC. bb10 fusions were tested in both soluble andplastic well-coated formats. PBMCs in complete RPMI medium were added toplates and incubated overnight. The cell culture supernatants were thenmeasured for TNFa and IL8 using ELISA. For two donor PBMC populations,bb10 fusions induce secretion of IL8 and TNF alpha at levels equal to orgreater than that of an agonistic anti-CD137 monoclonal antibody, URE1.

FIG. 8. In vivo stability is critical to the clinical efficacy of mostbiotherapeutics. Pharmacokinetic measurements of bb10 fusions wereperformed to assess the relative stability of DBDpp as compared to theantibody fusion partner. The in vivo stability was determined byanalysis of both the RSV and CD137 binding of the bi-specific antibodypresent in serum from CD1 mice that received a single intravenousinjection (1 mg/kg) of the fusion. Serum samples were collected at 15minutes and 48 hours, and were assayed by ELISA. Both N-terminal andC-terminal DBDpp fusion proteins demonstrate sustained stability invivo. As discussed in greater detail below, several embodiments involveDBDpp fusions with extended stability (e.g., on the order of 24 hours,48 hours, 72 hours, 96 hours, 6 days, 8 days, 10 days, or greater,including times between those listed).

FIG. 9. FIG. 9 depicts HPLC purification of a DBDpp produced accordingto several embodiments disclosed herein.

FIG. 10. FIG. 10 depicts SDS-PAGE analysis of purified DBDpp producedaccording to several embodiments disclosed herein. Lane 1 is a molecularweight marker, Lane 2 correspond to a purified DBDpp of SEQ ID NO: 58,and lanes 3-9 correspond to purified DBDpp of SEQ ID NOS: 51-57,respectively.

FIG. 11. FIG. 11 depicts a deconvoluted electrospray ionisation massspectrometry (ESI-MS) spectrum of SEQ ID NO. 54.

FIGS. 12A-12P. FIGS. 12A-12P depict data related to the binding ofCD137-targeting DBDpp to CD137 that was immobilized on a solid surface.FIGS. 12A, 12C, 12E, 12G, 12I, 12K, 12M, and 12O are sensorgrams forDBDpps of SEQ ID NOS: 51 (12A), 52 (12C), 53 (12E), 54 (12G), 55 (12I),56 (12K), 57 (12M) and 58 (12O). FIGS. 12B, 12D, 12F, 12H, 12J, 12L,12N, and 12P depict the corresponding steady state binding data forDBDpps of SEQ ID 51 (12B), 52 (12D), 53 (12F), 54 (12H), 55 (12J), 56(12L), 57 (12N) and 58 (12P).

FIG. 13. FIG. 13 depicts chromatographic data for the purification ofCD137 protein from Chinese Hamster Ovary (CHO) cell supernatant.

FIGS. 14A-14B. Analysis of proteins purified using DBDpp. FIG. 14Adepicts a Coomassie stained gel loaded with purified fractions fromDBDpp purification columns. Lane 1 is a molecular weight marker. Lane 2is IMAC-purified CD137 protein, and Lanes 3-8 are eluates from columnswith various DBDpp according to several embodiments herein. FIG. 14B isa western blot analysis with corresponding samples to those shown inFIG. 14A.

FIGS. 15A-15D. Thermal stability of DBDpp. FIG. 15A depicts assessmentof DR5 scFv binding to PD-L1PD-L1-Fc coated microplate wells afterexposure to various elevated temperatures. FIG. 15B depicts data showinga correlation between increased temperature and reduced PDL1 binding bya PD-L1PD-L1-directed scFv. FIG. 15C shows a DBDpp, according to oneembodiment disclosed herein (pb04 DBDpp), retained PD-L1PD-L1 bindingaffinity after being exposed to increasing temperatures, up to 100° C.FIG. 15D shows an additional DBDpp (pb06 DBDpp) that also demonstratesthermal stability and can bind PD-L1PD-L1 after being exposed totemperatures up to 100° C.

FIGS. 16A-16B. Cross-reactivity of DBDpp. FIG. 16A depicts data relatedto the ability of DBDpp to bind targets across species. In particular,FIG. 16A demonstrates that a soluble DBDpp directed against PD-L1 canbind to human PD-L1 (upper trace) as well as cynomolgus PD-L1 (lowertrace) with similar binding affinities. FIG. 16B depicts flow cytometrydata confirming that when expressed in a T cell, specifically a chimericantigen receptor T cell, the T cell can recognize and bind to both humanand cynomolgus PD-L1.

FIG. 17. Assessment of DBDpp-CAR expression and target binding. FIG. 17depicts data related to the DBDpp-CAR expression and CD-123-Fc bindingof various candidate DBDpp-CAR HEK-293T cells.

FIG. 18. DBDpp mediate signal transduction. FIG. 18 depicts data relatedto the expression and ability of DBDpp-CAR Jurkat cells to functionthrough an intracellular signaling pathway.

FIGS. 19A-19B. CD123-DBDpp-CAR T cells produce cytokines in response totarget binding. FIG. 19A shows data related to the production ofinterferon gamma (IFNγ) by T cells expressing DBDpp-CARs that targetCD123. FIG. 19B depicts similar data measuring the production ofinterleukin 2 (IL2) by CD123-targeting DBDpp-CAR T cells.

FIGS. 20A-20B. PD-L1-DBDpp-CAR T cells produce cytokines in response totarget binding. FIG. 20A shows data related to the production ofinterferon gamma (IFNγ) by T cells expressing DBDpp-CARs that targetPD-L1. FIG. 20B depicts similar data measuring the production ofinterleukin 2 (IL2) by PD-L1-targeting DBDpp-CAR T cells.

FIG. 21. CD123-DBDpp-CAR T cells proliferate in response to targetbinding. FIG. 21 depicts data related to the proliferation ofCD123-targeting DBDpp-CAR T cells as compared to control andCD123-targeting scFv.

FIG. 22. PD-L1-DBDpp-CAR T cells proliferate in response to targetbinding. FIG. 22 depicts data related to the proliferation ofPD-L1-targeting DBDpp-CAR T cells as compared to mock conditions.

FIGS. 23A-23B. T cells expressing DBDpp-CARs do not undergo excessiveexhaustion to a greater degree than scFv. FIG. 23A depicts expression ofthree exhaustion markers (LAG-3, PD-1, and TIM3) on T cells expressingvarious DBDpp-CARs at similar levels of the expression of those markerson scFv. FIG. 23B shows flow cytometry data depicting similar exhaustionmarker expression on DBDpp-CAR T cells (expressing CD123 targeting cg06DBDpp) as compared to a CAR T cell expressing CD123-specific scFv(32716).

FIGS. 24A-24D. T cells expressing DBDpp-CARs degranulate in response totarget binding. FIG. 24A depicts CD107a production (as a marker ofdegranulation of the DBDpp-CAR T cells) when CD123-targeting DBDpp-CAR Tcells are cultured alone. FIG. 24B shows CD107a production whenDBDpp-CAR T cells are co-cultured with CD123 negative K562 tumor cells.FIG. 24C shows CD107a when CD123-targeting DBDpp-CAR T cells areco-cultured with CD123 positive BDCM cells. FIG. 24D depicts data fromexperimental replicates of co-culture of CD123-targeting DBDpp-CAR Twith CD123 positive BDCM cells.

FIGS. 25A-25D. T cells expressing PD-L1-DBDpp-CARs degranulate inresponse to target binding. FIG. 25A shows CD107a expression (as amarker of degranulation of the DBDpp-CAR T cells) when PD-L1-targetingDBDpp-CAR T cells are cultured alone, e.g., unactivated. FIG. 25B showsthe measurement of CD107a when DBDpp-CAR T cells are co-cultured withPD-L1 negative K562 tumor cells. FIG. 25C shows increased CD107a whenPD-L1-targeting DBDpp-CAR T cells are co-cultured with PD-L1 positiveSUDHL1 cells. FIG. 25D depicts data from experimental replicates ofco-culture of PD-L1-targeting DBDpp-CAR T with PD-L1 positive SUDHL1cells.

FIGS. 26A-26D. T cells expressing DBDpp-CARs mediate target-specifictumor cytotoxicity. FIG. 26A shows data related CD123 targetingDBDpp-CAR T cells kill percentage of K562 tumor cells that are negativefor CD123. FIG. 26B shows kill percentages when the CD123 targetingDBDpp-CAR T cells are co-cultured with CD123 positive BDCM cells. Thedata from FIGS. 26A and 26B were generated using T cells from a firstdonor blood sample. FIGS. 26C and 26D show similar data from T cellscollected from a second donor.

FIGS. 27A-27F. T cells expressing DBDpp-CARs mediate target-specifictumor cytotoxicity. FIG. 27A shows data related to PD-L1 targetingDBDpp-CAR T cells kill percentage of K562 tumor cells that are negativefor PD-L1. The CAR T cells expressing the various PD-L1 targeting DBDppexhibited kill rates lower than mock controls. Similar data is shown inFIGS. 27C and 27E for two additional donors. FIG. 27B shows elevatedkill percentages when the PD-L1 targeting DBDpp-CAR T cells areco-cultured with PD-L1 positive SUDHL1 cells. Similar data is shown inFIGS. 27D and 27F for two additional donors.

FIGS. 28A-28D. DBDpp having reduced immunogenicity potential. Becausethe DBDpp as disclosed herein are synthetic, an analysis was performedto identify potentially immunogenic epitopes. A three-dimensional modelof a DBDpp (cg06) is shown in FIG. 28A. FIG. 28B depicts cg06 with one(of three) of the potentially immunogenic epitopes modified to be lesspotentially immunogenic. FIG. 28C depicts cg06 with two (of three) ofthe potentially immunogenic epitopes modified. FIG. 28D depicts cg06with all three of the potentially immunogenic epitopes modified.

FIGS. 29A-29B. DBDpp with modified epitopes retain functionality. FIG.29A depicts data related to CAR T cells expressing variants of CD123targeting DBDpp (cg06). Even with all three potentially immunogenicepitopes removed from the DBDpp sequence, the variants retain theability mediate signal transduction (activating Jurkat cells engineeredto express luciferase) after binding to CD123 positive BDCM target cells(unmodified cg06 designated with arrow). FIG. 29B shows similar efficacywhen modified variants bound to CD123 positive KG-1a cells (unmodifiedcg06 designated with arrow).

FIGS. 30A-30B. Dual marker expression on tumor cells. FIG. 30A depictsflow cytometry data for expression of CD123 on K562 cells, KG1a cells,BDCM cells, SUDHL cells, or H460 cells. FIG. 30B depicts flow cytometrydata for expression of PD-L1 on the same cells lines.

FIGS. 31A-31E. Bi-specific DBDpp-CAR T cells. FIG. 31A shows thepercentage of T cells expressing CD123 targeting DBDpp-CARs. FIG. 31Bshows the percentage of T cells expressing PD-L1 targeting DBDpp-CARs.FIG. 31C shows the percentage of T cells expressing bi-specificCD123-PD-L1 targeting DBDpp-CARs (expressed with cg06 DBDpp distal tothe T cell membrane versus the pb04 DBDpp). FIG. 31D shows thepercentage of T cells expressing bi-specific PD-L1-CD123 targetingDBDpp-CARs (expressed with pb04 DBDpp distal to the T cell membraneversus the cg06 DBDpp). FIG. 31E depicts data related to the increasedintracellular signaling of bispecific DBDpp.

FIG. 32. Competitive DBDpp Binding Assay. FIG. 32 demonstrates oneembodiment of a competitive binding assay that can be used to identifyDBDpp that display shared epitope binding even though the DBDpp testedhave different primary amino acid sequences.

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes onlyand are not to be construed as in any way limiting of the subject matterdescribed.

Definition of Terms

It is understood that wherever embodiments are described herein with thelanguage “comprising” otherwise analogous embodiments described in termsof “consisting of” and/or “consisting essentially of” are also provided.However, when used in the claims as transitional phrases, each should beinterpreted separately and in the appropriate legal and factual context(e.g., “comprising” is considered more of an open-ended phrase while“consisting of” is more exclusive and “consisting essentially of”achieves a middle ground).

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both A and B; A or B; A (alone); and B (alone).Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C”is intended to encompass each of the following embodiments: A, B, and C;A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A(alone); B (alone); and C (alone).

The terms “protein” and “polypeptide” are used interchangeably herein torefer to a biological polymer comprising units derived from amino acidslinked via peptide bonds; a protein can be composed of two or morepolypeptide chains.

The terms “antibody” or “immunoglobulin,” as used interchangeablyherein, include whole antibodies and antibody fragments including anyfunctional domain of an antibody such as an antigen-binding fragment orsingle chains thereof, an effector domain, salvage receptor bindingepitope, or portion thereof. A typical antibody comprises at least twoheavy (H) chains and two light (L) chains interconnected by disulfidebonds. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as VH) and a heavy chain constant region. The heavychain constant region is comprised of three domains, CH1, CH2, and CH3.Each light chain is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The lightchain constant region is comprised of one domain, C1. The VH and VLregions can be further subdivided into regions of hypervariablity,termed Complementarity Determining Regions (CDRs), interspersed withregions that are more conserved, termed framework regions (FW). Each VHand VL is composed of three CDRs and four FWs, arranged fromamino-terminus to carboxy-terminus in the following order: FW1, CDR1,FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies can mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system. Non-limiting types of antibodies ofthe present disclosure include typical antibodies, scFvs, andcombinations thereof where, for example, a DBDpp is covalently linked(e.g., via peptide bonds or via a chemical linker) to the N-terminus ofeither the heavy chain and/or the light chain of a typical whole(full-length) antibody, or intercalated in the H chain and/or the Lchain of a whole antibody.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to any functional domain of an antibody such as anantigen-binding fragment or single chains thereof, an effector domain ora portion thereof, and a salvage receptor binding epitope or a portionthereof. Examples of antibody fragments include, but are not limited to,Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, single chainantibodies, and multi-specific antibodies formed from antibodyfragments. “Antibody fragment” as used herein comprises anantigen-binding site or epitope binding site. In one embodiment, theDBDpp fusion protein comprises an effector domain or portion thereof. Inone embodiment, the DBDpp fusion protein comprises a salvage receptorbinding epitope, or portion thereof.

As used herein, the term, “Fc region” or simply “Fc” is understood tomean the carboxyl-terminal portion of an immunoglobulin chain constantregion, preferably an immunoglobulin heavy chain constant region, or aportion thereof. For example, an immunoglobulin Fc region may comprise(1) a CH1 domain, a CH2 domain, and a CH3 domain, (2) a CH1 domain and aCH2 domain, (3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3domain, or (5) a combination of two or more domains and animmunoglobulin hinge region. In a preferred embodiment theimmunoglobulin Fc region comprises at least an immunoglobulin hingeregion a CH2 domain and a CH3 domain, and preferably lacks the CH1domain. In one embodiment, the class of immunoglobulin from which theheavy chain constant region is derived is IgG (Igγ) (γ subclasses 1, 2,3, or 4). Other classes of immunoglobulin, IgA (Iga), IgD (Igδ), IgE(Igε) and IgM (Igμ), may be used. The choice of appropriateimmunoglobulin heavy chain constant region is discussed in detail inU.S. Pat. Nos. 5,541,087, and 5,726,044, each of which is incorporatedby reference herein, in their entirety. The choice of particularimmunoglobulin heavy chain constant region sequences from certainimmunoglobulin classes and subclasses to achieve a particular result isconsidered to be within the level of skill in the art. The portion ofthe DNA construct encoding the immunoglobulin Fc region preferablycomprises at least a portion of a hinge domain, and preferably at leasta portion of a CH3 domain of Fc gamma or the homologous domains in anyof IgA, IgD, IgE, or IgM. Furthermore, it is contemplated thatsubstitution or deletion of amino acids within the immunoglobulin heavychain constant regions may be useful in the practice of the methods andcompositions disclosed herein. One example would be to introduce aminoacid substitutions in the upper CH2 region to create an Fc variant withreduced affinity for Fc receptors (Cole, J. Immunol. 159:3613 (1997)).

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis (or other cytotoxic effects) of the target cell. To assessADCC activity of a molecule of interest, any in vitro ADCC assay knownin the art can be used, such as that described in U.S. Pat. No.5,500,362 or 5,821,337. Useful effector cells for such assays include,but are not limited to, peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest can be assessed in vivo, e.g., in an animalmodel such as that disclosed in Clynes et al. PNAS 95:652-656 (1998).

The terms “single chain variable fragment(s),” or “scFv” antibodies asused herein refer to forms of antibodies (e.g., antibody fragments)comprising the variable regions of only the heavy and light chains,connected by a linker peptide. In one embodiment, a DBDpp fusion proteincomprises a DBDpp and a scFv.

The term “linker” refers to a peptide or other chemical linkage locatedbetween a DBDpp and another polypeptide of a DBDpp fusion protein.Suitable linkers for coupling the two or more linked DBDpp will be clearto the persons skilled in the art and non-limiting examples aredescribed herein.

The term “operably linked,” as used herein, indicates that two moleculesare attached so as to each retain at least some level of functionalactivity that each molecule had alone (assuming that each molecule had afunction activity). In embodiments when one molecule was withoutfunctional activity, it is operably lined with another molecule if theother molecule retains at least some level of its functional activity.Operably linked can also refer to linkage of two non-function molecules.Two molecules can be “operably linked” whether they are attacheddirectly or indirectly (e.g., via a linker).

The terms “specifically binds” or “having selective affinity for” meanthat a binding agent such as a DBDpp reacts or associates morefrequently, more rapidly, with greater duration, with greater affinity,or with some combination of the above to the epitope, protein, or targetmolecule than with alternative substances, including proteins unrelatedto the target epitope. Because of the sequence identity betweenhomologous proteins in different species, specific binding can, inseveral embodiments, include a binding agent that recognizes a proteinor target in more than one species. Likewise, because of homology withincertain regions of polypeptide sequences of different proteins, specificbinding can include a binding agent that recognizes more than oneprotein or target. It is understood that, in certain embodiments, abinding agent that specifically binds a first target may or may notspecifically bind a second target. As such, “specific binding” does notnecessarily require (although it can include) exclusive binding, e.g.,binding to a single target. Thus, a binding agent may, in certainembodiments, specifically bind more than one target. In certainembodiments, multiple targets may be bound by the same antigen-bindingsite on the binding agent.

“Target” refers to any molecule or combination of molecules that can bebound by a DBDpp such as a DBDpp fusion protein, or other component ofthe DBDpp fusion protein such as an antibody or antibody variable domainfragment.

The terms “epitope” and “antigenic determinant” are used interchangeablyherein and refer to that portion of any molecule (e.g., a target ofinterest) capable of being recognized and specifically bound by aparticular binding agent (e.g., an DBDpp or antibody). When therecognized molecule is a polypeptide, epitopes can be formed fromcontiguous amino acids and noncontiguous amino acids and/or otherchemically active surface groups of molecules (such as carbohydrates)juxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained upon protein denaturing,whereas epitopes formed by tertiary folding are typically lost uponprotein denaturing. An epitope typically includes at least 3 aminoacids, and more usually, at least 5 or 8-10 amino acids in a uniquespatial conformation.

A “peptide tag” as used herein refers to a peptide sequence that is partof or attached (for instance through genetic engineering) to anotherprotein, to provide a function to the resultant fusion. Peptide tags areusually relatively short in comparison to a protein to which they arefused; by way of example, peptide tags are, in several embodiments, fouror more amino acids in length, such as, 5, 6, 7, 8, 9, 10, 15, 20, or 25or more amino acids. In some embodiments, the DBDpp is a fusion proteinthat contains a peptide tag. In other embodiments, the DBDppspecifically binds a peptide tag. Numerous peptide tags that have usesas provided herein are known in the art. Examples of peptide tags thatmay be a component of a DBDpp fusion protein or a target bound by aDBDpp (e.g., a DBDpp fusion protein). Examples of peptide tags that maybe a component of a DBDpp fusion protein or a target bound by a DBDpp(e.g., a DBDpp fusion protein) include but are not limited to HA(hemagglutinin), c-myc, the Herpes Simplex virus glycoprotein D (gD),T7, GST, GFP, MBP, Strep-tags, His-tags, Myc-tags, TAP-tags and FLAG®tag (Eastman Kodak, Rochester, N.Y.) Likewise, antibodies to the tagepitope allow detection and localization of the fusion protein in, forexample, affinity purification, Western blots, ELISA assays, andimmunostaining of cells.

The term “naturally occurring” when used in connection with biologicalmaterials such as a nucleic acid molecules, polypeptides, and hostcells, refers to those which are found in nature and not modified by ahuman being. Conversely, “non-natural” or “synthetic” when used inconnection with biological materials refers to those which are not foundin nature and have been modified by a human being.

As used herein “modifications” with respect to the sequence of referencescaffold SEQ ID NO:1 (or with respect to other sequences) includessubstitutions, deletions insertions and/or additions of the sequence ofthe corresponding amino acid position of SEQ ID NO:1 (or with respect tothe corresponding position of the other sequence).

A “substitution” with respect to the sequence of reference scaffold SEQID NO:1 (or with respect to other sequences) refers to a replacement ofa particular amino acid residue with a different amino acid residue at acorresponding amino acid position of SEQ ID NO:1 (or with respect to thecorresponding position of the other sequence).

A “conservative” amino acid substitution is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine(K), arginine (R), histidine (H)), acidic side chains (e.g., asparticacid (D), glutamic acid (E)), uncharged polar side chains (e.g., glycine(G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine(Y), cysteine (C)), nonpolar side chains (e.g., alanine (A), valine (V),leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine(M), tryptophan (W), beta-branched side chains (e.g., threonine (T),valine (V), isoleucine (I)) and aromatic side chains (e.g., tyrosine(Y), phenylalanine (F), tryptophan (W), histidine (H)). For example,substitution of a phenylalanine for a tyrosine is a conservativesubstitution. In one embodiment, conservative substitutions in thesequences of the DBDpp result in the specific binding of the DBDppcontaining the substitution to the target of interest to which it binds.In one embodiment, conservative substitutions in the sequences of theDBDpp do not abrogate the binding of the DBDpp containing thesubstitution to the target of interest to which it binds. Methods ofidentifying nucleotide and amino acid conservative substitutions andnon-conservative substitutions which confer, alter or maintain selectivebinding affinity are known in the art (see, e.g., Brummell, Biochem.32:1180-1187 (1993); Kobayashi, Protein Eng. 12(10):879-884 (1999); andBurks, PNAS 94:412-417 (1997)).

A “non-conservative” amino acid substitution is one in which one aminoacid residue is replaced with another amino acid residue having adissimilar side chain. In one embodiment, non-conservative substitutionsin the sequences of the DBDpp result in the specific binding of theDBDpp containing the substitution to the target of interest to which itbinds. In one embodiment, non-conservative substitutions in thesequences of the DBDpp do not abrogate the binding of the DBDppcontaining the substitution to the target of interest to which it binds.

“Non natural amino acids,” “amino acid analogs” and “non-standard aminoacid residues” are used interchangeably herein. Non-natural amino acidsthat can be substituted in a DBDpp as provided herein are known in theart. In one embodiment the non-natural amino acid is 4-hydroxyprolinewhich can be substituted for proline; 5-hydroxylysine which can besubstituted for lysine; 3-methylhistidine which can be substituted forhistidine; homoserine which can be substituted for serine; and ornithinewhich can be substituted for lysine. Additional examples of non-naturalamino acids that can be substituted in a DBDpp include, but are notlimited to molecules such as: D-isomers of the common amino acids,2,4-diaminobutyric acid, alpha-amino isobutyric acid, A-aminobutyricacid, Abu, 2-amino butyric acid, gamma-Abu, epsilon-Ahx, 6-aminohexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, beta-alanine, lanthionine,dehydroalanine, γ-aminobutyric acid, selenocysteine and pyrrolysinefluoro-amino acids, designer amino acids such as beta-methyl aminoacids, C alpha-methyl amino acids, and N alpha-methyl amino acids, orcombinations of non-natural amino acids. Still additional non-naturalamino acids can include 4-amino butyric acid,4-amino-3-hydroxy-5-phenylpentanoic acid,4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine, and/orD-isomers of amino acids. As discussed herein, in several embodimentsnon-natural amino acids or amino acid analogs can include deletion ofone or more amino acids from a sequence.

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. These terms include, but arenot limited to, DNA, RNA, cDNA (complementary DNA), mRNA (messengerRNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (smallnuclear RNA), snoRNA (short nucleolar RNA), miRNA (microRNA), genomicDNA, synthetic DNA, synthetic RNA, and/or tRNA.

The term “naked DNA” as used herein refers to DNA (e.g., histone freeDNA) encoding a protein such as a DBDpp (e.g., a CAR) is a DNA cloned ina suitable expression vector in proper orientation for expression (e.g.,a plasmid). Viral vectors which may be used include but are not limitedto SIN lentiviral vectors, retroviral vectors, foamy virus vectors,adenovirus vectors, adeno-associated virus (AAV) vectors, hybrid vectorsand/or plasmid transposons (for example sleeping beauty transposonsystem) or integrase based vector systems. Other vectors that can beused in connection with making and using DBDpp are described herein orotherwise known in the art.

The terms “vector”, “cloning vector” and “expression vector” as usedherein refer to the vehicle by which a nucleic acid sequence (e.g., aDBDpp coding sequence) can be maintained or amplified in a host cell(e.g., cloning vector) or introduced into a host cell, so as totransform the host and promote expression (e.g., transcription andtranslation) of the introduced sequence. Vectors include plasmids,phages, viruses, etc.

A “host cell” includes an individual cell or cell culture which can beor has been a recipient of nucleic acids encoding a DBDpp. Host cellsincludes but are not limited to viral particles, phagemids, bacteria,yeast plant, animal, and mammalian cells. Host cells include progeny ofa single host cell, and the progeny may not necessarily be completelyidentical (in morphology or in total DNA complement) to the originalparent cell due to natural, accidental, or deliberate mutation and/orchange. A host cell includes cells transfected or infected in vivo, invitro, or ex vivo with nucleic acids encoding a DBDpp. In some examples,the host cell is capable of expressing and displaying DBDpp on itssurface, such as for example, in phage display. “Expression” includestranscription and/or translation.

A “library” of DBDpp refers to a plurality of unique DBDpp, andoptionally including multiple DBDpp that bind to the same target, butwith varied binding sites and/or specificities.

A “vector library” of DBDpp refers to a plurality of unique nucleicacids encoding DBDpp (as above, optionally including nucleic acidsencoding DBDpp that bind to the same target, but with varied bindingsites and/or specificities).

As used herein, the terms “solid support,” “support,” “matrices,” and“resins” are used interchangeably and refer to, without limitation, anycolumn (or column material), bead, test tube, microtiter dish, solidparticle (for example, agarose or sepharose), microchip (for example,silicon, silicon-glass, or gold chip), or membrane (e.g., biologic orfilter membrane) to which a DBDpp, antibody, or other protein may beattached (e.g., coupled, linked, or adhered), either directly orindirectly (for example, through other binding partner intermediatessuch as other antibodies or Protein A), or in which a DBDpp or antibodymay be embedded (for example, through a receptor or channel). Reagentsand techniques for attaching polypeptides to solid supports (e.g.,matrices, resins, plastic, etc.) are well known in the art. Suitablesolid supports include, but are not limited to, a chromatographic resinor matrix (e.g., SEPHAROSE-4 FF agarose beads), the wall or floor of awell in a plastic microtiter dish, a silica based biochip,polyacrylamide, agarose, silica, nitrocellulose, paper, plastic, nylon,metal, and combinations thereof. DBDpp and other compositions may beattached on a support material by a non-covalent association or bycovalent bonding, using reagents and techniques known in the art. In oneembodiment, the DBDpp is coupled to a chromatography material using alinker.

As used herein, the terms “pharmaceutically acceptable,” or“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a human without the production oftherapeutically prohibitive undesirable physiological effects such asnausea, dizziness, gastric upset and the like.

“Modulate,” means adjustment or regulation of amplitude, frequency,degree, or activity. In another related aspect, such modulation may bepositively modulated (e.g., an increase in frequency, degree, oractivity) or negatively modulated (e.g., a decrease in frequency,degree, or activity). In several embodiments, modulation in a positiveor negative direction is referenced as compared to the cell, tissue, ororgan function prior to administration of a therapeutic. In additionalembodiments, modulation in a positive or negative direction isreferenced with respect to a normal, healthy cell, tissue or organ.

An “effective amount” of a DBDpp such as a DBDpp fusion protein asprovided herein is an amount sufficient to carry out a specificallystated purpose such as to bring about an observable change in the levelof one or more biological activities related to the target to which theDBDpp (e.g., a DBDpp fusion protein) binds. In certain embodiments, thechange increases the level of target activity. In other embodiments, thechange decreases the level of target activity. An “effective amount” canbe determined empirically and in a routine manner, in relation to thestated purpose. The term “therapeutically effective amount” refers to anamount of a DBDpp such as a DBDpp fusion protein, or other therapeuticagent effective to “treat” (e.g., reduce symptoms of) a disease ordisorder in a subject (mammal). A “prophylactically effective amount”refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired prophylactic result.

“Patient,” “subject,” “animal” and “mammal” are used interchangeably andrefer to mammals such as human patients and non-human primates, as wellas experimental animals such as rabbits, rats, and mice, and otheranimals. Animals include all vertebrates, e.g., mammals and non-mammals,such as chickens, amphibians, and reptiles. “Mammal” as used hereinrefers to any member of the class Mammalia, including, withoutlimitation, humans and nonhuman primates such as chimpanzees and otherapes and monkey species; farm animals such as cattle, sheep, pigs, goatsand horses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs, and the like. In aparticular embodiment, the patient is a human. The term does not denotea particular age or sex. Thus, adult and newborn subjects, as well asembryos and fetuses, whether male or female, are intended to be includedwithin the scope of this term.

The terms “treat,” “treatment,” and “treating,” as used herein refer toboth therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow down (lessen or delay) thesymptoms, complications, or biochemical indicia of a disease, condition,or disorder, alleviating the symptoms or arresting or inhibiting furtherdevelopment of the disease, condition, or disorder. Treatment can beprophylactic (to prevent or delay the onset of the disease, or toprevent the manifestation of clinical or subclinical symptoms thereof)or therapeutic suppression or alleviation of symptoms after themanifestation of the disease, condition, or disorder targeted pathologiccondition, prevent the pathologic condition, pursue or obtain beneficialresults, or lower the chances of the individual developing the conditioneven if the treatment is ultimately unsuccessful. Those in need oftreatment include those already with the condition as well as thoseprone to have the condition or those in whom the condition is to beprevented. Treatment can be with a DBDpp fusion protein alone or incombination with an additional therapeutic agent.

“Cancer,” “tumor,” or “malignancy” are used as synonymous terms andrefer to any of a number of diseases that are characterized byuncontrolled, abnormal proliferation of cells, the ability of affectedcells to spread locally or through the bloodstream and lymphatic systemto other parts of the body (metastasize) as well as any of a number ofcharacteristic structural and/or molecular features. “Tumor,” as usedherein refers to all neoplastic cell growth and proliferation, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues. A “cancerous tumor,” or “malignant cell” is understood as acell having specific structural properties, lacking differentiation andbeing capable of invasion and metastasis. Cancers that can be treatedusing DBDpp fusion proteins provided herein include without limitation,breast, lung, brain, bone, liver, kidney, colon, head and neck, ovarian,hematopoietic (e.g., leukemia), and prostate cancer. Other types ofcancer and tumors that may be treated using DBDpp-containing antibodiesare described herein or otherwise known in the art.

The terms tumor antigen or cancer antigen are used interchangeablyherein. Tumor and cancer antigens may be tumor-specific antigen (TSA),cancer-specific antigens (CSA) tumor-associated antigen (TAA) orcancer-associated antigens (CAA). A TSA is an antigen that is unique totumor cells and does not occur on other cells in the body. A TAA is anantigen that is found on both tumor and some normal cells. Because ofthe dynamic nature of tumors, in some instances, tumor cells may expressunique antigens at certain stages, and at others also express antigensthat are also expressed on non-tumor cells. Thus, inclusion of a certainmarker as a TAA does not preclude it being considered a TSA. Examples ofTAAs and TSAs that may be specifically bound by a DBDpp include but arenot limited to: CD19, CD20, CD22, ROR 1, mesothelin, CD33/1L3Ra, cMet,PSMA, Glycolipid F77, EGFRvIII, GD2, NY-ESO-1TCR, MAGE A3 TCR MARTI,gp100 (Pmel 17), tyrosinase, TRP1, TRP2, MAGE1, MAGE3, BAGE, GAGE1,GAGE2, pi5, CEA; p53, Ras, HER-2/neu; BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK,MYL-RAR; EBVA, HPV antigens E6 and E7, TSP-180, MAGE4, MAGE5, MAGE6,RAGE, NY-ESO, p185erbB2, p180erbB3, nm-23H1, PSA, CA 19-9, CA72-4, CAM17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p15, p16, 43-9F,5T4(791Tgp72) alpha-fetoprotem, beta-HCG, BCA225, BTAA, CA125, CA15-3\CA 27.29\BCAA, CA195, CA242, CA50, CAM43, CD68\I, CO-029, FGF5,G250, Ga733VEpCAM, HTgp-175, M344, MA50, MG7-Ag, MOV 18, NB/70K,NY-CO-1, RCAS1, SDCCAG16, TA90\Mac-2, TAAL6, TAG72, TLP, and TPS.

The term “target cell” as used herein refers to cells which are involvedin a disease and can be targeted by DBDpp containing compositions. Othertarget cells include any cell in a subject (e.g., a human or animal)that can be targeted by DBDpp of the invention. The target cell can be acell expressing or overexpressing a target specifically bound by a DBDppfusion protein.

The term “effector cells” are leukocytes which express one or more FcRsand perform effector functions. Preferably, the cells express at leastFc(RIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred in certain embodiments. Theeffector cells can be isolated from native source thereof, e.g., fromblood or PBMCs as described herein or otherwise known in the art. In aspecific embodiment, the effector cells are human effector cells.

The term “effector function” refers to the specialized immune functionof a differentiated cell. Effector function of a T-cell, for example,may be cytolytic activity or helper activity including the secretion ofcytokines.

The terms “T-cell” and “T-lymphocyte” are interchangeable and usedsynonymously herein. Examples include but are not limited to naive Tcells, central memory T cells, effector memory T cells or combinationsthereof.

The term “immune cell” as used herein refers to the cells of themammalian immune system including but not limited to antigen presentingcells, B-cells, basophils, cytotoxic T-cells, dendritic cells,eosinophils, granulocytes, helper T-cells, leukocytes, lymphocytes,macrophages, mast cells, memory cells, monocytes, natural killer cells,neutrophils, phagocytes, plasma cells and T-cells.

The term “immune response” as used herein refers to immunities includingbut not limited to innate immunity, humoral immunity, cellular immunity,immunity, inflammatory response, acquired (adaptive) immunity,autoimmunity and/or overactive immunity.

The term “transduction” as used herein refers to the introduction of aforeign nucleic acid into a cell using a viral vector. “Transfection” asused herein refers to the introduction of a foreign nucleic acid into acell using recombinant DNA technology. The term “transformation” meansthe introduction of a “foreign” (e.g., extrinsic, extracellular, orotherwise non-endogenous) nucleic acid (DNA or RNA) sequence to a hostcell, so that the host cell will express the introduced nucleic acid toproduce a desired substance, such as a protein or enzyme coded by theintroduced coding sequence. The introduced nucleic acid sequence canalso be called a “cloned” or “foreign” gene or sequence, can includeregulatory or control sequences, such as start, stop, promoter, signal,secretion, or other sequences used by a cell's genetic machinery. Thenucleic acid sequence can include nonfunctional sequences or sequenceswith no known function. A host cell that receives and expressesintroduced nucleic acid (e.g., DNA or RNA) has been “transformed” and isa “transformant” or a “clone.” The DNA or RNA introduced to a host cellcan come from any source, including cells of the same genus or speciesas the host cell, or cells of a different genus or species or may benon-naturally occurring.

“Cell surface receptor” refers to molecules and complexes of moleculescapable of receiving a signal and the transmission of such a signalacross the plasma membrane of a cell. An example of a cell surfacereceptor provided herein is an activated integrin receptor, for example,an activated αvβ3 integrin receptor on a metastatic cell. As usedherein, “cell surface receptor” also includes a molecule expressed on acell surface that contains a DBDpp capable of binding a target ofinterest. The term “receptor” denotes a cell-associated protein thatbinds to, or otherwise interacts with, a molecule (e.g., a ligand) andmediates the effect of the ligand on the cell. In several embodiments,the molecule that interacts with a receptor is a bioactive molecule.Membrane-bound cell-surface receptors are characterized by amulti-domain structure comprising an extracellular ligand-bindingdomain, a membrane spanning domain, and an intracellular effector domainthat is typically involved in signal transduction.

“Chimeric antigen receptor” or “CAR” or “CARs” as used herein refers toengineered receptors, which graft an antigen or target specificity ontocells (for example T cells such as naive T cells, central memory Tcells, effector memory T cells, NK cells, NKT cells or combinationthereof). CARs are also known as artificial T-cell receptors, chimericT-cell receptors or chimeric immunoreceptors.

De Novo Binding Domain Polypeptides

The terms “de novo binding domain” and DBD are used interchangeablyherein to describe a target binding sequence sharing certain sequenceand certain structural features of the reference scaffold sequence:MGSWAEFKQRLAAIKTRLEALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:1). The terms DBDpp and DBD polypeptides includesingular (i.e., a DBD polypeptide) and plural (i.e., DBD polypeptides)references unless otherwise indicated explicitly or by context. A DBDppis polypeptide that can specifically (non-randomly) bind to a targetmolecule.

It has been discovered, and is disclosed herein in several embodiments,that a non-naturally occurring and targetless (Applicant has noknowledge of a target that can be bound) antiparallel three-helicalbundle having the amino acid sequence of SEQ ID NO:1 can be used as areference scaffold platform for producing de novo binding domain (DBD)containing polypeptides (DBDpp) that bind to a target of interest andfor creating libraries of DBDpp which can be screened for DBDpp havingdesired functional and/or biological activities. Accordingly, in someaspects, the disclosure relates to the use of DBDpp, in methods ofproducing DBDpp having desired properties such as the ability to bind atarget of interest; methods of producing libraries of DBDpp; thelibraries of DBDpp produced by such methods; methods for screening suchlibraries of DBDpp for desired biological activities; and the DBDppidentified from such libraries.

Unless otherwise indicated, the practice of the disclosed compositionsand methods employs standard techniques of molecular biology (includingrecombinant techniques, tissue culture, and cell transformation),microbiology, cell biology, biochemistry and immunology, which arewithin the skill of the art. Such techniques are typically performedaccording to the manufacturer's specifications or as commonlyaccomplished using or routinely modifying known procedures such as,those set forth in Sambrook et al. (Molecular Cloning: A LaboratoryManual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989)); PCR Technology: Principles and Applications for DNAAmplification (ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992);Oligonucleotide Synthesis (Gait, ed., 1984); Animal Cell Culture(Freshney, ed., 1987); Handbook of Experimental Immunology (Weir et al.,eds.; Gene Transfer Vectors for Mammalian Cells (Miller, ed., 1987);Current Protocols in Molecular Biology (Ausubel., ed., 1987); PCRProtocols: A Guide to Methods and Applications (Innis, ed., AcademicPress, San Diego, Calif., 1990); Mattila, et al., Nucleic Acids Res.19:967 (1991); Eckert, et al., PCR Methods and Applications 1:17 (1991);PCR (McPherson, ed., IRL Press, Oxford); PCR: The Polymerase ChainReaction, (Mullis, ed., 1994); Harlow, Antibodies: A Laboratory Manual,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988) and Kontermann, ed.,“The Antibody Engineering Lab Manual” (Springer Verlag, Heidelberg/NewYork, 2000); Current Protocols in Immunology (Coligan, ed., 1991); TheImmunoassay Handbook (Wild, ed., Stockton Press NY, 1994); and Methodsof Immunological Analysis (Masseyeff., ed., Weinheim: VCH Verlagsgesellschaft mbH, 1993); and Gennaro, et al. 2000, Remington: theScience and Practice of Pharmacy, 20th Ed. Lipincott Williams andWilkins: Baltimore, Md., or as described herein. Unless specificdefinitions are provided, the nomenclature utilized in connection with,and the laboratory procedures and techniques of analytical chemistry,synthetic organic chemistry, and medicinal and pharmaceutical chemistrydescribed herein, are those known and used in the art. Additionally,standard techniques can be used for chemical syntheses, chemicalanalyses, recombinant production, purification, pharmaceuticalpreparation, formulation, delivery, and treatment of patients.

In one embodiment, the DBDpp is not derived from a natural cellularligand of public record (as of the filing of U.S. ProvisionalApplication Ser. No. 62/143,772, filed Apr. 6, 2015 and according to theApplicant's knowledge). In another embodiment, the DBDpp is not derivedfrom an immunoglobulin-derived antigen binding domain, or anotherantibody domain such as a constant region, a variable region, acomplementarity determining region (CDR), a framework region, an Fcdomain, or a hinge region. In another embodiment, the DBDpp does notcontain three CDRs. In another embodiment, the DBDpp does not containCDR1 and CDR2. In yet another embodiment, the DBDpp does not containCDR1. In yet another embodiment, the DBDpp does not contain CDR2. Inanother embodiment, the DBDpp in not derived from protein A. In anotherembodiment the DBDpp is not derived from a natural bacterial receptor.In another embodiment the DBDpp is not derived from fibronectin. Inanother embodiment the DBDpp is not derived from fibronectin type IIIdomain. In yet another embodiment, the DBDpp is not derived from aknottin protein. In yet another embodiment, the DBDpp is not derivedfrom a lipocalin. In yet another embodiment, the DBDpp is not derivedfrom an affibody.

Sequence Characteristics

As indicated above, the reference scaffold polypeptide of SEQ ID NO:1contains three anti-parallel alpha helices and is a variant of anon-naturally occurring and targetless polypeptide sequence originallyengineered as an exercise in protein folding. Provided herein are DBDppcontaining certain modifications of amino acid residues in the sequenceof reference scaffold polypeptide of SEQ ID NO:1 that confer the abilityof the DBDpp to bind a target of interest and the use of the DBDpp as atarget binding and targeting agent.

In one embodiment, an individual DBDpp has a length of about 65 to 150amino acids, about 65 to 125 amino acids, about 65 to 100 amino acids,about 65 to 90 amino acids, about 65 to 80 amino acids, about 65 to 70amino acids. It is also contemplated in some embodiments, that a DBDpphas a length of about 75 to 150 amino acids, about 75 to 125 aminoacids, about 75 to 100 amino acids, about 75 to 90 amino acids, about 75to 80 amino acids. DBDpp can be naked or conjugated to other molecules,including but not limited to, toxins and radioisotopes. In stilladditional embodiments, longer DBDpp are employed, for example DBDppranging in length from about 150 to about 160 amino acids, about 160 toabout 170 amino acids, about 170 to about 180 amino acids, about 180 toabout 190 amino acids, about 190 to about 200 amino acids, or any lengthbetween those listed (including endpoints).

For known binding proteins, the specific residues that constitute thebinding region of the molecule either have been (or theoretically canbe) experimentally determined. Natural binding proteins (e.g. antibodiesor protein A) have identifiable residues that promote the binding totheir known targets. However, unlike natural ligands and bindingproteins, the designed protein α3d (SEQ ID NO: 49) or the referencescaffold sequence of SEQ ID NO:1 are not known to specifically bind toanother protein (e.g., a target). Therefore endogenous binding residuescannot be utilized as a guide to engineer novel binding specificity. Inthe construction of DBDpp that bind to targets, residues were consideredfor mutation (e.g., randomization within the library) if they weresurface exposed—exhibiting significant solvent accessibility. Therelative accessibility of a residue within the domain (area D) ascompared to the isolated state (area I) is represented as a percentvalue (% A). Amino residues of SEQ ID NO:1 that have % A values lessthan about 10% to 11% (e.g., residues corresponding to F7, L11, I14,L18, L21, S24, L28, F31, I35, F38, L42, Y45, G49, V53, L56, A60, 163,and L67, of SEQ ID NO:1), are believed to be inaccessible to theexterior solvent and are considered to be interior core residues of theSEQ ID NO:1 structure. Conversely, amino acid residues of SEQ ID NO:1with % A values that are greater than about 10% to 11% are believed tooccupy positions that have greater potential for interaction a target ofinterest. Binding surfaces of proteins are typically composed of severalamino residues that are either adjacent, or in close proximity, to eachother in three-dimensional space. Therefore, a secondary considerationin the construction of libraries, according to several embodimentsherein, was the relative proximity of these selected residues within thepredicted secondary and tertiary structure of the DBDpp.

Protein secondary structure such as alpha helices can change dependingon environmental variables such as temperature, matrix or buffercomposition and concentration. The alpha helical secondary structures ofthe reference polypeptide sequence of SEQ ID NO:1 are predicted to becomposed of residues G2-A20 for helix 1, residues L28-A44 for helix 2,and residues E52-Y70 for helix 3. In additional embodiments, the alphahelical secondary structures of the reference polypeptide sequence ofSEQ ID NO:1 are predicted to be composed of residues W4-L21 for helix 1,residues E25-Y45 for helix 2, and residues P51-Y70 for helix 3. Theamino acid positions of the reference scaffold corresponding to alphahelical residues with low solvent accessibility are: F7, L11, I14, L18,L21, L28, F31, I35, F38, L42, Y45, V53, L56, A60, I63 and L67 of SEQ IDNO: 1. The amino acid positions of the reference scaffold correspondingto solvent accessible, alpha helical residues are: G2, S3, W4, A5, E6,K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34,A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59, A61,A62, R64, D65, E66, Q68, A69, and Y70 of SEQ ID NO:1. The amino acidpositions of the reference scaffold corresponding to the non-alphahelical residues are as follows: M1, G22, G23, S24, E25, A26, E27, K46,G47, K48, G49, N50, P51, R71, H72, and N73 of SEQ ID NO:1.

In one embodiment, DBDpp are defined as target-binding polypeptidescomposed of SEQ ID NO:1 with one or more amino acid substitutions. Inone embodiment, a sequence alignment of the DBDpp with SEQ ID NO:1 wouldreveal a sequence identity greater than 90%. In other embodiments, asequence alignment of the DBDpp with SEQ ID NO:1 would reveal a sequenceidentity greater than 80%. In other embodiments, a sequence alignment ofthe DBDpp with SEQ ID NO:1 would reveal a sequence identity greater than70%. In other embodiments, a sequence alignment of the DBDpp with SEQ IDNO:1 would reveal a sequence identity greater than 60%. In otherembodiments, a sequence alignment of the DBDpp with SEQ ID NO:1 wouldreveal a sequence identity greater than 50%.

In some embodiments, DBDpp residues with % A values that are less than10% would remain constant, or be substituted with a conserved amino acidchange. In particular embodiments, the solvent accessible (i.e., % Agreater than 10) residue DBDpp has an amino acid sequence that aremodified subject to mutagenesis would be located within regions of thepolypeptide associated with alpha-helical secondary structure. The alphahelical positions of the sequence of SEQ ID NO:1 having solventinaccessible residues correspond to F7, L11, I14, L18, L21, L28, F31,I35, F38, L42, Y45, V53, L56, A60, I63, and L67, of SEQ ID NO:1. Aminoacid substitutions in these positions are preferably conservative innature and can include unconventional or non-natural amino acids. Insome embodiments, the selection of natural amino acid substitutionsincludes L, I, V, A and F (and W, Y, M). In some DBDpp, the solventinaccessible residues of a DBD contained in a DBDpp is greater than 60%,70%, 80%, or 90%, or is 100% identical to the corresponding residues inSEQ ID NO:1. F7, L11, I14, L18, L21, L28, F31, I35, F38, L42, Y45, V53,L56, A60, I63, and L67, of SEQ ID NO:1.

In one embodiment, a DBDpp comprises an amino acid sequence of SEQ IDNO:1 wherein a total of 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5to 50, 5 to 55, or 5 to 60 amino acid residues have been modified; andwherein the DBDpp specifically binds a target of interest. In anotherembodiment, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to55, or 5 to 60 of the modified amino acid residues are substitutions. Inanother embodiment, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, or 5 to50 of the modified amino acid residues are conservative substitutions.In another embodiment, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, or 5to 50 of the modified amino acid residues are non-conservativesubstitutions. In a further embodiment, 5 to 15, 5 to 20, 5 to 25, 5 to30, 5 to 35, 5 to 40, or 5 to 45 of the amino acid residue modificationsare conservative substitutions and 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5to 35, 5 to 40, or 5 to 45 of the amino acid residue modifications arenon-conservative substitutions. In additional embodiments, 1 to 25, 1 to30, 1 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 of thesubstitutions are at amino acid residues of SEQ ID NO:1 selected fromthe group consisting of: Ml, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13,K15, T16, R17, E19, A20, L21, G22, G23, S24, E25, A26, E27, A29, A30,E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, Y45, K46, G47, K48,G49, N50, P51, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66,Q68, A69, Y70, R71, H72, and N73. In additional embodiments, 1 to 25, 1to 30, 1 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 of thesubstitutions are at amino acid residues of SEQ ID NO:1 selected fromthe group consisting of: Ml, G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13,K15, T16, R17, E19, A20, G22, G23, S24, E25, A26, E27, A29, A30, E32,K33, E34, A36, A37, E39, S40, E41, Q43, A44, K46, G47, K48, G49, N50,P51, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69,Y70, R71, H72, and N73. In a further embodiment, 1 to 20, 1 to 30, or 1to 40 of the substitutions are at amino acid residues of SEQ ID NO:1selected from the group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10,A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37,E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64,D65, E66, Q68, A69, and Y70. In an optional further embodiment, theDBDpp optionally further comprises an amino acid sequence wherein 1 to5, 1 to 10, 1 to 15, 5 to 10 or 5 to 15 of the residues corresponding tothe solvent inaccessible residues of the amino acid sequence of SEQ IDNO:1 are substituted and wherein the DBDpp specifically binds a targetof interest. In a further optional embodiment, the substituted residuescorresponding to a solvent inaccessible residue of SEQ ID NO:1 areselected from the group consisting of: F7, L11, I14, L18, L28, F31, I35,F38, L42, V53, L56, A60, I63, and L67, and Y70. In some embodiments, thesubstituted residues corresponding to a solvent inaccessible residue ofSEQ ID NO:1 are selected from the group consisting of: F7, L11, I14,L18, L21, L28, F31, I35, F38, L42, Y45, V53, L56, A60, I63, and L67, andY70. In an additional embodiment, the DBDpp is a fusion protein. In oneembodiment, the DBDpp is attached to a solid support. In a furtherembodiment, the solid support is selected from the group consisting of:a bead, a glass slide, a chip, a gelatin, and an agarose. In anadditional embodiment, the DBDpp specifically binds a target of interestselected from the group consisting of: a nucleic acid, anoligosaccharide, a peptide, a protein, a cell surface antigen, and asmall organic molecule. In a further embodiment, the DBDpp specificallybinds a protein selected from the group consisting of: animmunoglobulin, an enzyme, a hormone, a serum protein, a cell surfaceprotein, a therapeutic protein, a TSA, a CSA, and a protein containing apeptide tag. In another embodiment, the DBDpp specifically binds atarget disclosed herein. Nucleic acids encoding the DBDpp and vectorscontaining the nucleic acids are also provided. Host cells (includingviral particles) containing the nucleic acids and vectors are alsoprovided. In some embodiments, the host cell displays the DBDpp on itssurface. In additional embodiments, the host cell is a prokaryote or aeukaryote that display the DBDpp on its surface. In a furtherembodiment, the host cell is a phage that displays the DBDpp on itssurface. In a further embodiment, the host cell is a human immune cellthat expresses a DBDpp fusion protein on its surface. Librariescomprising a plurality of DBDpp are also provided.

In one embodiment, an isolated DBDpp comprises an amino acid sequencevariation of SEQ ID NO:1 wherein 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5to 35, 5 to 40 solvent accessible amino acid residues of SEQ ID NO:1 aresubstituted, and wherein 1 to 5, 1 to 10, 1 to 15, 5 to 10 or 5 to 15solvent inaccessible residues of SEQ ID NO:1 are optionally substitutedby a conservative amino acid substitution, and wherein the DBDppspecifically binds a target of interest. In some embodiments, thesubstituted solvent accessible amino acid residues of SEQ ID NO:1 have a% A of greater than 10. In some embodiments, the substituted solventinaccessible amino acid residues of SEQ ID NO:1 have a % A of less than10. In one embodiment, the substituted solvent accessible amino acidresidues of SEQ ID NO:1 are selected from the group consisting of: G2,S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29,A30, E32, K33, E34, A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55,R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, and Y70. In someembodiments, at least 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 35 of thesolvent accessible acid residues are substituted. In some embodiments,at least 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 of the solventaccessible acid residues of SEQ ID NO:1 are substituted withconservative amino acid residue substitutions. In some embodiments, atleast 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 of the solvent accessibleacid residues of SEQ ID NO:1 are substituted with non-conservative aminoacid residue substitutions. In some embodiments, the amino acidsubstitutions do not contain proline. In some embodiments, the aminoacid substitutions do not contain cysteine or proline. In someembodiments, the amino acid residue substitutions include no more thanone cysteine. In some embodiments, 1 to 5, 1 to 10, 5 to 15, 5 to 20, 5to 25, 5 to 30, or 5 to 35 of the solvent accessible acid residues aresubstituted. In some embodiments, 5 to 41, 10 to 41, 15 to 41, 20 to 41,25 to 41, 30 to 41, or 35 to 41 of the solvent accessible acid residuesare substituted. In some embodiments, 5 to 35 of the solvent accessibleacid residues are substituted with conservative substitutions and 5 to35 of the solvent accessible acid residues are substituted withnon-conservative substitutions, or 5 to 25 of the solvent accessibleacid residues are substituted with conservative substitutions and 5 to25 of the solvent accessible acid residues are substituted withnon-conservative substitutions. In an optional further embodiment, theDBDpp optionally further comprises an amino acid sequence wherein 1 to5, 1 to 10, 1 to 15, 5 to 10 or 5 to 15 of the residues corresponding tothe solvent inaccessible residues of the amino acid sequence of SEQ IDNO:1 are substituted and wherein the DBDpp specifically binds a targetof interest. In a further optional embodiment, the substituted residuescorresponding to a solvent inaccessible residue of SEQ ID NO:1 areselected from the group consisting of: F7, L11, I14, L18, L28, F31, I35,F38, L42, V53, L56, A60, I63, and L67, and Y70. In a further optionalembodiment, the substituted residues corresponding to a solventinaccessible residue of SEQ ID NO:1 are selected from the groupconsisting of: F7, L11, I14, L18, L21, L28, F31, I35, F38, L42, Y45,V53, L56, A60, I63, and L67, and Y70. In an additional embodiment, theDBDpp is a fusion protein. In one embodiment, the DBDpp is attached to asolid support. In a further embodiment, the solid support is selectedfrom the group consisting of: a bead, a glass slide, a chip, a gelatin,and an agarose. In an additional embodiment, the DBDpp specificallybinds a target of interest selected from the group consisting of: anucleic acid, an oligosaccharide, a peptide, a protein, a cell surfaceantigen, and a small organic molecule. In a further embodiment, theDBDpp specifically binds a protein selected from the group consistingof: an immunoglobulin, an enzyme, a hormone, a serum protein, a cellsurface protein, a therapeutic protein, a TSA, a CSA, and a proteincontaining a peptide tag. In another embodiment, the DBDpp specificallybinds a target disclosed herein. Nucleic acids encoding the DBDpp andvectors containing the nucleic acids are also provided. Host cells(including viral particles) containing the nucleic acids and vectors arealso provided. In some embodiments, the host cell displays the DBDpp onits surface. In additional embodiments, the host cell is a prokaryote ora eukaryote that display the DBDpp on its surface. In a furtherembodiment, the host cell is a phage that displays the DBDpp on itssurface. In a further embodiment, the host cell is a human immune cellthat expresses a DBDpp fusion protein on its surface. Librariescomprising a plurality of DBDpp are also provided.

The term “loop” refers to sequences in the DBD corresponding to the looplocated between, for example, helix 1 and helix 2 of reference scaffoldSEQ ID NO:1 (e.g., positions 22-24 of SEQ ID NOS:2-6, and Z₁ of SEQ IDNOS:7-11) and/or the loop located between helix 2 and helix 3 ofreference scaffold SEQ ID NO:1 e.g., positions 46-48 of SEQ ID NOS:2-6,and Z₂ of SEQ ID NOS:7-11). In particular embodiments, one or both ofthe Z₁ and Z₂ loops are amino acid sequences consisting of 2 to 5, 2 to10, 2 to 15, 2 to 20, 2 to 25 or 2 to 30 amino acid residues (includingendpoints and any number in between those listed). In some embodiments,one or both of the Z₁ and Z₂ loops are amino acid sequences consistingof 1, 2, 3, 4, 5, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20,or more than 20 amino acid residues (including endpoints and any numberin between those listed). In a further embodiment, at least 50%, 60%,70%, 80%, 90%, or 95% of the amino acid residues of the Z₁ and/or Z₂loop are glycine or serine. In additional embodiments, at least 50%,60%, 70%, 80%, 90%, or 95% of the amino acid residues of the Z₁ and/orZ₂ loop are selected from the group consisting of glycine, serine,threonine, alanine, proline, histidine, asparagine, aspartic acid,glutamine, glutamic acid, lysine and arginine. In one embodiment the Z₁loop has the amino acid sequence GGS. In one embodiment the Z₂ loop hasthe amino acid sequence KGKG.

In one embodiment, a DBDpp comprises an amino acid sequence ofMGSWX₅X₆FK X₉X₁₀LAX13IKX₁₆X₁₇LEALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2), wherein X₅, X₆, X₉, X₁₀, X₁₃, X₁₆,X₁₇, X₃₀, X₃₃, X₃₄, X₃₇, X₄₀, X₄₁, and X₄₄, is a natural and/ornon-natural amino acid residue, and wherein the DBDpp specifically bindsa target of interest. In an additional embodiment, X_(n) is a naturalamino acid residue. In a further embodiment, X_(n) is a natural aminoacid residue other than cysteine or proline. In a particular embodiment,the DBDpp does not contain the amino acid sequence LAAIKTRLQ (SEQ IDNO:50). In an additional embodiment, the DBDpp is a fusion protein. Insome embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of thesubstituted amino acid residues of SEQ ID NO:1 are substituted withconservative amino acid residue substitutions. In some embodiments, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residuesof SEQ ID NO:1 are substituted with non-conservative amino acid residuesubstitutions. In some embodiments, the amino acid substitutions do notcontain proline. In some embodiments, the amino acid substitutions donot contain cysteine. In some embodiments, neither proline nor cysteineis included in the amino acid substitutions. In some embodiments, theamino acid residue substitutions include no more than one cysteine. Insome embodiments, 1 to 12 of the solvent accessible acid residues aresubstituted with conservative substitutions and 1 to 12 of the solventaccessible acid residues are substituted with non-conservativesubstitutions, or 5 to 12 of the solvent accessible acid residues aresubstituted with conservative substitutions and 5 to 12 of the solventaccessible amino acid residues are substituted with non-conservativesubstitutions. In another embodiment, the DBDpp specifically binds atarget of interest selected from the group consisting of: a nucleicacid, an oligosaccharide, a peptide, a protein, a cell surface antigen,and a small organic molecule. In a further embodiment, the DBDppspecifically binds a protein selected from the group consisting of: animmunoglobulin, an enzyme, a hormone, a serum protein, a cell surfaceprotein, a therapeutic protein, a TSA, a CSA, and a protein containing apeptide tag. In a further embodiment, the DBDpp specifically binds atarget disclosed herein. In an additional embodiment, a librarycontaining a plurality of DBDpp is provided. Nucleic acids encoding theDBDpp and vectors containing the nucleic acids are also provided. Hostcells (including viral particles) containing the nucleic acids andvectors are also provided. In some embodiments, the host cell is aprokaryote or a eukaryote that display the DBDpp on its surface. In someembodiments, the host cell displays the DBDpp on its surface. In afurther embodiment, the host cell is a phage that displays the DBDpp onits surface. In a further embodiment, the host cell is a human immunecell that expresses a DBDpp fusion protein on its surface. In oneembodiment, the DBDpp is attached to a solid support. In a furtherembodiment, the solid support is selected from the group consisting of:a bead, a glass slide, a chip, a gelatin, and an agarose.

In one embodiment, the DBDpp comprises an amino acid sequence ofMGSWAEFKQRLAAIKTRLEALGGSEAELAAFX32X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN(SEQ ID NO:3), wherein X₃₂, X₃₃, X₃₆, X₃₉, X₄₀, X₄₃, X₅₇, X₅₈, X₆₁, X₆₄,X₆₅, and X₆₈, is a natural and/or non-natural amino acid residue, andwherein the DBDpp specifically binds a target of interest. In anadditional embodiment, X_(n) is a natural amino acid residue. In afurther embodiment, X_(n) is a natural amino acid residue other thancysteine or proline. In a particular embodiment, the DBDpp does notcontain the amino acid sequence LAAIKTRLQ (SEQ ID NO:50). In anadditional embodiment, the DBDpp is a fusion protein. In someembodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of thesubstituted amino acid residues of SEQ ID NO:1 are substituted withconservative amino acid residue substitutions. In some embodiments, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residuesof SEQ ID NO:1 are substituted with non-conservative amino acid residuesubstitutions. In some embodiments, the amino acid substitutions do notcontain proline. In some embodiments, the amino acid substitutions donot contain cysteine. In some embodiments, neither proline nor cysteineis included in the amino acid substitutions. In some embodiments, theamino acid residue substitutions include no more than one cysteine. Insome embodiments, 1 to 12 of the solvent accessible acid residues aresubstituted with conservative substitutions and 1 to 12 of the solventaccessible acid residues are substituted with non-conservativesubstitutions, or 5 to 12 of the solvent accessible acid residues aresubstituted with conservative substitutions and 5 to 12 of the solventaccessible amino acid residues are substituted with non-conservativesubstitutions. In another embodiment, the DBDpp specifically binds atarget of interest selected from the group consisting of: a nucleicacid, an oligosaccharide, a peptide, a protein, a cell surface antigen,and a small organic molecule. In a further embodiment, the DBDppspecifically binds a protein selected from the group consisting of: animmunoglobulin, an enzyme, a hormone, a serum protein, a cell surfaceprotein, a therapeutic protein, a TSA, a CSA, and a protein containing apeptide tag. In a further embodiment, the DBDpp specifically binds atarget disclosed herein. In an additional embodiment, a librarycontaining a plurality of DBDpp is provided. Nucleic acids encoding theDBDpp and vectors containing the nucleic acids are also provided. Hostcells (including viral particles) containing the nucleic acids andvectors are also provided. In some embodiments, the host cell is aprokaryote or a eukaryote that display the DBDpp on its surface. In someembodiments, the host cell displays the DBDpp on its surface. In afurther embodiment, the host cell is a phage that displays the DBDpp onits surface. In a further embodiment, the host cell is a human immunecell that expresses a DBDpp fusion protein on its surface. In oneembodiment, the DBDpp is attached to a solid support. In a furtherembodiment, the solid support is selected from the group consisting of:a bead, a glass slide, a chip, a gelatin, and an agarose.

In one embodiment, the DBDpp comprises an amino acid sequence of MGSWXSEFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN(SEQ ID NO:4), wherein X₅, X₈, X₉, X₁₂, X₁₅, X₁₆, X₁₉, X₅₅, X₅₈, X₅₉,X₆₂, X₆₅, and X₆₆ is a natural and/or non-natural amino acid residue,and wherein the DBDpp specifically binds a target of interest. In anadditional embodiment, X_(n) is a natural amino acid residue. In afurther embodiment, X_(n) is a natural amino acid residue other thancysteine or proline. In a particular embodiment, the DBDpp does notcontain the amino acid sequence LAAIKTRLQ (SEQ ID NO:50). In anadditional embodiment, the DBDpp is a fusion protein. In someembodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of thesubstituted amino acid residues of SEQ ID NO:1 are substituted withconservative amino acid residue substitutions. In some embodiments, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residuesof SEQ ID NO:1 are substituted with non-conservative amino acid residuesubstitutions. In some embodiments, the amino acid substitutions do notcontain proline. In some embodiments, the amino acid substitutions donot contain cysteine. In some embodiments, neither proline nor cysteineis included in the amino acid substitutions. In some embodiments, theamino acid residue substitutions include no more than one cysteine. Insome embodiments, 1 to 12 of the solvent accessible acid residues aresubstituted with conservative substitutions and 1 to 12 of the solventaccessible acid residues are substituted with non-conservativesubstitutions, or 5 to 12 of the solvent accessible acid residues aresubstituted with conservative substitutions and 5 to 12 of the solventaccessible amino acid residues are substituted with non-conservativesubstitutions. In another embodiment, the DBDpp specifically binds atarget of interest selected from the group consisting of: a nucleicacid, an oligosaccharide, a peptide, a protein, a cell surface antigen,and a small organic molecule. In a further embodiment, the DBDppspecifically binds a protein selected from the group consisting of: animmunoglobulin, an enzyme, a hormone, a serum protein, a cell surfaceprotein, a therapeutic protein, a TSA, a CSA, and a protein containing apeptide tag. In a further embodiment, the DBDpp specifically binds atarget disclosed herein. In an additional embodiment, a librarycontaining a plurality of DBDpp is provided. Nucleic acids encoding theDBDpp and vectors containing the nucleic acids are also provided. Hostcells (including viral particles) containing the nucleic acids andvectors are also provided. In some embodiments, the host cell is aprokaryote or a eukaryote that display the DBDpp on its surface. In someembodiments, the host cell displays the DBDpp on its surface. In afurther embodiment, the host cell is a phage that displays the DBDpp onits surface. In a further embodiment, the host cell is a human immunecell that expresses a DBDpp fusion protein on its surface. In oneembodiment, the DBDpp is attached to a solid support. In a furtherembodiment, the solid support is selected from the group consisting of:a bead, a glass slide, a chip, a gelatin, and an agarose.

In one embodiment, the DBDpp comprises an amino acid sequence ofMGSWXSX₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN (SEQ ID NO:5), wherein X₅, X₆, X₉, X₁₀, X₁₃,X₁₆, X₁₇, X₃₂, X₃₃, X₃₆, X₃₉, X₄₀, X₄₃, X₅₅, X₅₈, X₅₉, X₆₂, X₆₅, andX₆₆, is a natural and/or non-natural amino acid residue, and wherein theDBDpp specifically binds a target of interest. In an additionalembodiment, X_(n) is a natural amino acid residue. In a furtherembodiment, X_(n) is a natural amino acid residue other than cysteine orproline. In a particular embodiment, the DBDpp does not contain theamino acid sequence LAAIKTRLQ (SEQ ID NO:50). In an additionalembodiment, the DBDpp is a fusion protein. In some embodiments, at least1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the substituted amino acid residuesof SEQ ID NO:1 are substituted with conservative amino acid residuesubstitutions. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 of the above amino acid residues of SEQ ID NO:1 are substitutedwith non-conservative amino acid residue substitutions. In someembodiments, the amino acid substitutions do not contain proline. Insome embodiments, the amino acid substitutions do not contain cysteine.In some embodiments, neither proline nor cysteine is included in theamino acid substitutions. In some embodiments, the amino acid residuesubstitutions include no more than one cysteine. In some embodiments, 1to 12 of the solvent accessible acid residues are substituted withconservative substitutions and 1 to 12 of the solvent accessible acidresidues are substituted with non-conservative substitutions, or 5 to 12of the solvent accessible acid residues are substituted withconservative substitutions and 5 to 12 of the solvent accessible aminoacid residues are substituted with non-conservative substitutions. Inanother embodiment, the DBDpp specifically binds a target of interestselected from the group consisting of: a nucleic acid, anoligosaccharide, a peptide, a protein, a cell surface antigen, and asmall organic molecule. In a further embodiment, the DBDpp specificallybinds a protein selected from the group consisting of: animmunoglobulin, an enzyme, a hormone, a serum protein, a cell surfaceprotein, a therapeutic protein, a TSA, a CSA, and a protein containing apeptide tag. In a further embodiment, the DBDpp specifically binds atarget disclosed herein. In an additional embodiment, a librarycontaining a plurality of DBDpp is provided. Nucleic acids encoding theDBDpp and vectors containing the nucleic acids are also provided. Hostcells (including viral particles) containing the nucleic acids andvectors are also provided. In some embodiments, the host cell is aprokaryote or a eukaryote that display the DBDpp on its surface. In someembodiments, the host cell displays the DBDpp on its surface. In afurther embodiment, the host cell is a phage that displays the DBDpp onits surface. In a further embodiment, the host cell is a human immunecell that expresses a DBDpp fusion protein on its surface. In oneembodiment, the DBDpp is attached to a solid support. In a furtherembodiment, the solid support is selected from the group consisting of:a bead, a glass slide, a chip, a gelatin, and an agarose.

In one embodiment, the DBDpp comprises an amino acid sequence of MGSWXSEFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN (SEQ ID NO:6), wherein X₅, X₈, X₉, X₁₂,X₁₅, X₁₆, X₁₉, X₃₀, X₃₃, X₃₄, X₃₇, X₄₀, X₄₁, X₄₄, X₅₇, X₅₈, X₆₁, X₆₄,X₆₅, and X₆₈, is a natural and/or non-natural amino acid residue, andwherein the DBDpp specifically binds a target of interest. In anadditional embodiment, X_(n) is a natural amino acid residue. In afurther embodiment, X_(n) is a natural amino acid residue other thancysteine or proline. In a particular embodiment, the DBDpp does notcontain the amino acid sequence LAAIKTRLQ (SEQ ID NO:50). In anadditional embodiment, the DBDpp is a fusion protein. In someembodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of thesubstituted amino acid residues of SEQ ID NO:1 are substituted withconservative amino acid residue substitutions. In some embodiments, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residuesof SEQ ID NO:1 are substituted with non-conservative amino acid residuesubstitutions. In some embodiments, the amino acid substitutions do notcontain proline. In some embodiments, the amino acid substitutions donot contain cysteine. In some embodiments, neither proline nor cysteineis included in the amino acid substitutions. In some embodiments, theamino acid residue substitutions include no more than one cysteine. Insome embodiments, 1 to 12 of the solvent accessible acid residues aresubstituted with conservative substitutions and 1 to 12 of the solventaccessible acid residues are substituted with non-conservativesubstitutions, or 5 to 12 of the solvent accessible acid residues aresubstituted with conservative substitutions and 5 to 12 of the solventaccessible amino acid residues are substituted with non-conservativesubstitutions. In another embodiment, the DBDpp specifically binds atarget of interest selected from the group consisting of: a nucleicacid, an oligosaccharide, a peptide, a protein, a cell surface antigen,and a small organic molecule. In a further embodiment, the DBDppspecifically binds a protein selected from the group consisting of: animmunoglobulin, an enzyme, a hormone, a serum protein, a cell surfaceprotein, a therapeutic protein, a TSA, a CSA, and a protein containing apeptide tag. In a further embodiment, the DBDpp specifically binds atarget disclosed herein. In an additional embodiment, a librarycontaining a plurality of DBDpp is provided. Nucleic acids encoding theDBDpp and vectors containing the nucleic acids are also provided. Hostcells (including viral particles) containing the nucleic acids andvectors are also provided. In some embodiments, the host cell is aprokaryote or a eukaryote that display the DBDpp on its surface. In someembodiments, the host cell displays the DBDpp on its surface. In afurther embodiment, the host cell is a phage that displays the DBDpp onits surface. In a further embodiment, the host cell is a human immunecell that expresses a DBDpp fusion protein on its surface. In oneembodiment, the DBDpp is attached to a solid support. In a furtherembodiment, the solid support is selected from the group consisting of:a bead, a glass slide, a chip, a gelatin, and an agarose.

Also provided is an isolated DBDpp that comprises an amino acid sequenceof:MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7), wherein X₅, X₆, X₉, X₁₀, X₁₃, X₁₆,X₁₇, X₂₈, X₃₁, X₃₂, X₃₅, X₃₈, X₃₉, and X₄₂, is a natural and/ornon-natural amino acid residue, wherein Z₁ and Z₂ are 2 to 30 naturaland/or non-natural amino acid residues, and wherein the DBDppspecifically binds a target of interest. In an additional embodiment,X_(n) is a natural amino acid residue. In a further embodiment, X_(n) isa natural amino acid residue other than cysteine or proline. In aparticular embodiment, the DBDpp does not contain the amino acidsequence LAAIKTRLQ (SEQ ID NO:50). In an additional embodiment, theDBDpp is a fusion protein. In some embodiments, at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 of the substituted amino acid residues of SEQ ID NO:1are substituted with conservative amino acid residue substitutions. Insome embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the aboveamino acid residues of SEQ ID NO:1 are substituted with non-conservativeamino acid residue substitutions. In some embodiments, the amino acidsubstitutions do not contain proline. In some embodiments, the aminoacid substitutions do not contain cysteine. In some embodiments, neitherproline nor cysteine is included in the amino acid substitutions. Insome embodiments, the amino acid residue substitutions include no morethan one cysteine. In some embodiments, 1 to 12 of the solventaccessible acid residues are substituted with conservative substitutionsand 1 to 12 of the solvent accessible acid residues are substituted withnon-conservative substitutions, or 5 to 12 of the solvent accessibleacid residues are substituted with conservative substitutions and 5 to12 of the solvent accessible amino acid residues are substituted withnon-conservative substitutions. In another embodiment, the DBDppspecifically binds a target of interest selected from the groupconsisting of: a nucleic acid, an oligosaccharide, a peptide, a protein,a cell surface antigen, and a small organic molecule. In a furtherembodiment, the DBDpp specifically binds a protein selected from thegroup consisting of: an immunoglobulin, an enzyme, a hormone, a serumprotein, a cell surface protein, a therapeutic protein, a TSA, a CSA,and a protein containing a peptide tag. In a further embodiment, theDBDpp specifically binds a target disclosed herein. In an additionalembodiment, a library containing a plurality of DBDpp is provided.Nucleic acids encoding the DBDpp and vectors containing the nucleicacids are also provided. Host cells (including viral particles)containing the nucleic acids and vectors are also provided. In someembodiments, the host cell is a prokaryote or a eukaryote that displaythe DBDpp on its surface. In some embodiments, the host cell displaysthe DBDpp on its surface. In a further embodiment, the host cell is aphage that displays the DBDpp on its surface. In a further embodiment,the host cell is a human immune cell that expresses a DBDpp fusionprotein on its surface. In one embodiment, the DBDpp is attached to asolid support. In a further embodiment, the solid support is selectedfrom the group consisting of: a bead, a glass slide, a chip, a gelatin,and an agarose.

Also provided is an isolated DBDpp that comprises an amino acid sequenceof: MGSWAEFKQRLAAIKTRLEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN (SEQ ID NO:8), wherein X₃₀, X₃₁, X₃₄,X₃₇, X₃₈, X₄₁, X₅₂, X₅₃, X₅₆, X₅₉, X₆₀, and X₆₃, is a natural and/ornon-natural amino acid residue, wherein Z₁ and Z₂ are 2 to 30 naturaland/or non-natural amino acid residues, and wherein the DBDppspecifically binds a target of interest. In an additional embodiment,X_(n) is a natural amino acid residue. In a further embodiment, X_(n) isa natural amino acid residue other than cysteine or proline. In aparticular embodiment, the DBDpp does not contain the amino acidsequence LAAIKTRLQ (SEQ ID NO:50). In an additional embodiment, theDBDpp is a fusion protein. In some embodiments, at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 of the substituted amino acid residues of SEQ ID NO:1are substituted with conservative amino acid residue substitutions. Insome embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the aboveamino acid residues of SEQ ID NO:1 are substituted with non-conservativeamino acid residue substitutions. In some embodiments, the amino acidsubstitutions do not contain proline. In some embodiments, the aminoacid substitutions do not contain cysteine. In some embodiments, neitherproline nor cysteine is included in the amino acid substitutions. Insome embodiments, the amino acid residue substitutions include no morethan one cysteine. In some embodiments, 1 to 12 of the solventaccessible acid residues are substituted with conservative substitutionsand 1 to 12 of the solvent accessible acid residues are substituted withnon-conservative substitutions, or 5 to 12 of the solvent accessibleacid residues are substituted with conservative substitutions and 5 to12 of the solvent accessible amino acid residues are substituted withnon-conservative substitutions. In another embodiment, the DBDppspecifically binds a target of interest selected from the groupconsisting of: a nucleic acid, an oligosaccharide, a peptide, a protein,a cell surface antigen, and a small organic molecule. In a furtherembodiment, the DBDpp specifically binds a protein selected from thegroup consisting of: an immunoglobulin, an enzyme, a hormone, a serumprotein, a cell surface protein, a therapeutic protein, a TSA, a CSA,and a protein containing a peptide tag. In a further embodiment, theDBDpp specifically binds a target disclosed herein. In an additionalembodiment, a library containing a plurality of DBDpp is provided.Nucleic acids encoding the DBDpp and vectors containing the nucleicacids are also provided. Host cells (including viral particles)containing the nucleic acids and vectors are also provided. In someembodiments, the host cell is a prokaryote or a eukaryote that displaythe DBDpp on its surface. In some embodiments, the host cell displaysthe DBDpp on its surface. In a further embodiment, the host cell is aphage that displays the DBDpp on its surface. In a further embodiment,the host cell is a human immune cell that expresses a DBDpp fusionprotein on its surface. In one embodiment, the DBDpp is attached to asolid support. In a further embodiment, the solid support is selectedfrom the group consisting of: a bead, a glass slide, a chip, a gelatin,and an agarose.

Also provided is an isolated DBDpp that comprises an amino acid sequenceMGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN (SEQ ID NO:10), wherein X₅, X₆, X₉,X₁₀, X₁₃, X₁₆, X₁₇, X₃₀, X₃₁, X₃₄, X₃₇, X₃₈, X₄₁, X₅₀, X₅₃, X₅₄, X₅₇,X₆₀, and X₆₁ is a natural and/or non-natural amino acid residue, whereinZ₁ and Z₂ are 2 to 30 natural and/or non-natural amino acid residues,and wherein the DBDpp specifically binds a target of interest. In anadditional embodiment, X_(n) is a natural amino acid residue. In afurther embodiment, X_(n) is a natural amino acid residue other thancysteine or proline. In a particular embodiment, the DBDpp does notcontain the amino acid sequence LAAIKTRLQ (SEQ ID NO:50). In anadditional embodiment, the DBDpp is a fusion protein. In someembodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of thesubstituted amino acid residues of SEQ ID NO:1 are substituted withconservative amino acid residue substitutions. In some embodiments, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residuesof SEQ ID NO:1 are substituted with non-conservative amino acid residuesubstitutions. In some embodiments, the amino acid substitutions do notcontain proline. In some embodiments, the amino acid substitutions donot contain cysteine. In some embodiments, neither proline nor cysteineis included in the amino acid substitutions. In some embodiments, theamino acid residue substitutions include no more than one cysteine. Insome embodiments, 1 to 12 of the solvent accessible acid residues aresubstituted with conservative substitutions and 1 to 12 of the solventaccessible acid residues are substituted with non-conservativesubstitutions, or 5 to 12 of the solvent accessible acid residues aresubstituted with conservative substitutions and 5 to 12 of the solventaccessible amino acid residues are substituted with non-conservativesubstitutions. In another embodiment, the DBDpp specifically binds atarget of interest selected from the group consisting of: a nucleicacid, an oligosaccharide, a peptide, a protein, a cell surface antigen,and a small organic molecule. In a further embodiment, the DBDppspecifically binds a protein selected from the group consisting of: animmunoglobulin, an enzyme, a hormone, a serum protein, a cell surfaceprotein, a therapeutic protein, a TSA, a CSA, and a protein containing apeptide tag. In a further embodiment, the DBDpp specifically binds atarget disclosed herein. In an additional embodiment, a librarycontaining a plurality of DBDpp is provided. Nucleic acids encoding theDBDpp and vectors containing the nucleic acids are also provided. Hostcells (including viral particles) containing the nucleic acids andvectors are also provided. In some embodiments, the host cell is aprokaryote or a eukaryote that display the DBDpp on its surface. In someembodiments, the host cell displays the DBDpp on its surface. In afurther embodiment, the host cell is a phage that displays the DBDpp onits surface. In a further embodiment, the host cell is a human immunecell that expresses a DBDpp fusion protein on its surface. In oneembodiment, the DBDpp is attached to a solid support. In a furtherembodiment, the solid support is selected from the group consisting of:a bead, a glass slide, a chip, a gelatin, and an agarose.

Also provided is an isolated DBDpp that comprises an amino acid sequenceofMGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEAL X₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN (SEQ ID NO:11), wherein X₅, X₈, X₉,X₁₂, X₁₅, X₁₆, X₁₉, X₂₈, X₃₁, X₃₂, X₃₅, X₃₈, X₃₉, X₄₂, X₅₂, X₅₃, X₅₆,X₅₉, X₆₀, and X₆₃, is a natural and/or non-natural amino acid residue,wherein Z₁ and Z₂ are 2 to 30 natural and/or non-natural amino acidresidues, and wherein the DBDpp specifically binds a target of interest.In an additional embodiment, X_(n) is a natural amino acid residue. In afurther embodiment, X_(n) is a natural amino acid residue other thancysteine or proline. In a particular embodiment, the DBDpp does notcontain the amino acid sequence LAAIKTRLQ (SEQ ID NO:50). In anadditional embodiment, the DBDpp is a fusion protein. In someembodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of thesubstituted amino acid residues of SEQ ID NO:1 are substituted withconservative amino acid residue substitutions. In some embodiments, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above amino acid residuesof SEQ ID NO:1 are substituted with non-conservative amino acid residuesubstitutions. In some embodiments, the amino acid substitutions do notcontain proline. In some embodiments, the amino acid substitutions donot contain cysteine. In some embodiments, neither proline nor cysteineis included in the amino acid substitutions. In some embodiments, theamino acid residue substitutions include no more than one cysteine. Insome embodiments, 1 to 12 of the solvent accessible acid residues aresubstituted with conservative substitutions and 1 to 12 of the solventaccessible acid residues are substituted with non-conservativesubstitutions, or 5 to 12 of the solvent accessible acid residues aresubstituted with conservative substitutions and 5 to 12 of the solventaccessible amino acid residues are substituted with non-conservativesubstitutions. In another embodiment, the DBDpp specifically binds atarget of interest selected from the group consisting of: a nucleicacid, an oligosaccharide, a peptide, a protein, a cell surface antigen,and a small organic molecule. In a further embodiment, the DBDppspecifically binds a protein selected from the group consisting of: animmunoglobulin, an enzyme, a hormone, a serum protein, a cell surfaceprotein, a therapeutic protein, a TSA, a CSA, and a protein containing apeptide tag. In a further embodiment, the DBDpp specifically binds atarget disclosed herein. In an additional embodiment, a librarycontaining a plurality of DBDpp is provided. Nucleic acids encoding theDBDpp and vectors containing the nucleic acids are also provided. Hostcells (including viral particles) containing the nucleic acids andvectors are also provided. In some embodiments, the host cell is aprokaryote or a eukaryote that display the DBDpp on its surface. In someembodiments, the host cell displays the DBDpp on its surface. In afurther embodiment, the host cell is a phage that displays the DBDpp onits surface. In a further embodiment, the host cell is a human immunecell that expresses a DBDpp fusion protein on its surface. In oneembodiment, the DBDpp is attached to a solid support. In a furtherembodiment, the solid support is selected from the group consisting of:a bead, a glass slide, a chip, a gelatin, and an agarose.

In some embodiments, the DBDpp comprises a substitution at acorresponding position in the sequence of SEQ ID NO:1 selected from thegroup consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15,T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41,Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68,A69, and Y70. In additional embodiments, the DBDpp comprisessubstitutions of at least 1, 5, 10, 15, 20, or 30 of the above positionsin the sequence of SEQ ID NO:1. These substitutions can be conservative,non-conservative, or a mix of conservative and non-conservativesubstitutions. In some embodiments, the substitutions do not include theaddition of a proline or cysteine. In some embodiments, thesubstitutions include no more than a single cysteine. In some DBDpp,these residues may be greater than 90% identical to SEQ ID NO:1. Inother DBDpp, these residues may be greater than 80% identical to SEQ IDNO:1. In other DBDpp, these residues may be greater than 70% identicalto SEQ ID NO:1. In other DBDpp, these residues may be greater than 60%identical to SEQ ID NO:1. In other DBDpp, these residues may be greaterthan 50% identical to SEQ ID NO:1. In other DBDpp, these residues may begreater than 40% identical to SEQ ID NO:1. In other DBDpp, theseresidues may be greater than 30% identical to SEQ ID NO:1. In otherDBDpp, these residues are greater than 20% identical to SEQ ID NO:1. Inother DBDpp, these residues are greater than 10% identical to SEQ IDNO:1.

In some embodiments, the DBDpp comprises a substitution at a position inthe sequence of SEQ ID NO:1 selected from the group consisting of: M1,L21, G22, G23, S24, E25, A26, E27, Y45, K46, G47, K48, G49, N50, P51,R71, H72, and N73.

Additionally provided herein are DBDpp in which amino acid residues havebeen deleted from the amino terminus, the carboxy terminus or both theamino and carboxy termini of the corresponding sequence of SEQ ID NO: 1.In some embodiments the DBDpp contains a sequence with 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 amino acid residues deleted from the aminoterminus of the DBDpp sequence corresponding to the sequence of SEQ IDNO: 1. In some embodiments the DBDpp contains a sequence with 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues deleted from thecarboxy terminus of the DBDpp sequence corresponding to the sequence ofSEQ ID NO:1. In some embodiments the DBDpp contains a sequence with 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11, or 12 amino acid residues deletedfrom the amino terminus of the corresponding sequence of SEQ ID NO:1 anda sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acidresidues deleted from the carboxy terminus corresponding to the sequenceof SEQ ID NO:1. In additional embodiments, the DBDpp contains a sequencewith 1-5, 1-10, or 1 to 15 amino acid residues deleted from the carboxyterminus of the sequence corresponding to SEQ ID NO: 1. In someembodiments the DBDpp contains a sequence with 1-5, 1-10, or 1 to 15amino acid residues deleted from the amino terminus of the sequencecorresponding to the SEQ ID NO:1 and a sequence with 1-5, 1-10, or 1 to15 amino acid residues deleted from the carboxy terminus of the sequencecorresponding to SEQ ID NO:1.

In some embodiments, the DBDpp contains a sequence that differs from thecorresponding sequence in reference SEQ ID NO:1 in 2 or more categoriesof sequence modifications (i.e., substitutions, deletions, insertions,and additions). For example DBDpp may include combinations of amino aciddeletions, insertions and substitutions compared to the correspondingsequence in the reference polypeptide sequence. In some embodiments theDBDpp contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino aciddeletions within the sequence reference sequence shown in SEQ ID NO:1.In some embodiments the DBDpp contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore than 10 amino acid insertions within the reference sequence shownin SEQ ID NO:1.

DBDpp Bind to Targets of Interest

According to some embodiments, DBDpp can bind to a target of interest,and in several embodiments, have no discernable impact on the functionof the target. Alternatively, in several embodiments, DBDpp can bind toa target of interest and completely or partially inhibit, antagonize,agonize, block, increase, stimulate or interfere with the biologicalactivity of that target. Binding can be identified as agonistic orantagonistic and determined using or routinely modifying assays,bioassays, and/or animal models known in the art for evaluating suchactivity.

A DBDpp agonist refers to a DBDpp that in some way increases or enhancesthe biological activity of the DBDpp target or has biological activitycomparable to a known agonist of the DBDpp target. In anotherembodiment, the DBDpp is an antagonist of the target it binds. A DBDppantagonist refers to a DBDpp that completely or partially blocks or insome way interferes with the biological activity of the DBDpp targetprotein or has biological activity comparable to a known antagonist orinhibitor of the DBDpp target protein.

Expressions like “binding affinity for a target”, “binding to a target”and the like refer to a property of a polypeptide which may be directlymeasured through the determination of the affinity constants, e.g., theamount of DBDpp that associates and dissociates at a given antigenconcentration. Different methods can be used to characterize themolecular interaction, such as, but not limited to, competitionanalysis, equilibrium analysis and microcalorimetric analysis, andreal-time interaction analysis based on surface plasmon resonanceinteraction (for example using a Biacore® instrument). These methods arewell-known to the skilled person and are described, for example, in NeriD et al. (1996) Tibtech 14:465-470 and Jansson M et al. (1997) J BiolChem 272:8189-8197.

Affinity requirements for a given DBDpp binding event are contingent ona variety of factors including, but not limited to: the composition andcomplexity of the binding matrix, the valency and density of both theDBDpp and target molecules, and the functional application of the DBDpp.In one embodiment, DBDpp bind a target of interest with a dissociationconstant (KD) of less than or equal to 5×10⁻³ M, 10⁻³ M, 5×10 M, 10⁻⁴ M,5×10⁻⁵ M, or 10⁻⁵ M. In an additional embodiment, a DBDpp binds a targetof interest with a KD of less than or equal to 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸ M, or 10⁻⁸ M. In additional embodiments, a DBDpp bindsa target of interest with a KD less than or equal to 5×10⁻⁹ M, 10⁻⁹ M,5×10⁻¹° M, 10⁻¹° M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M,10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M. In severalembodiments, the DBDpp generated by the methods disclosed herein have adissociation constant selected from the group consisting of between 10⁻⁴M and 10⁻⁵ M, between 10⁻⁵ M and 10⁻⁶ M, between 10⁻⁶ M and 10⁻⁷ M,between 10⁻⁷ M and 10⁻⁸ M, between 10⁻⁸ M and 10⁻⁹ M, between 10⁻⁹ M and10⁻¹⁰ M, between 10⁻¹⁰ M and 10⁻¹¹ M and between 10⁻¹¹ M and 10⁻¹² M.

In one embodiment a DBDpp binds a target of interest in active form. Inone embodiment a DBDpp reversibly binds a target of interest in activeform and also releases the bound target in active form. In oneembodiment a DBDpp binds a target of interest in the native form. Inspecific embodiments, DBDpp bind targets of interest with off-rates orK_(off) of greater than or equal to 10⁻¹⁰ sec⁻¹, 5×10⁻⁹ sec⁻¹, 10⁻⁹sec⁻¹, 5×10⁻⁸ sec⁻¹, 10⁻⁸ sec⁻¹, 5×10⁻⁷ sec⁻¹, 10⁻⁷ sec⁻¹, 5×10⁻⁶ sec⁻¹,10⁻⁶ sec⁻¹, 5×10⁻⁵ sec⁻¹, 10⁻⁵ sec⁻¹, 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻³sec⁻¹, 10⁻³ sec⁻¹, 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻¹ sec⁻¹, or 10⁻¹sec⁻¹.

Binding experiments to determine KD and off-rates can be performed in anumber of conditions including, but not limited to, [pH 6.0, 0.01% Tween20], [pH 6.0, 0.1% gelatin], [pH5.0, 0.01% Tween 20], [pH9.0, 0.1% Tween20], [pH6.0, 15% ethylene glycol, 0.01% Tween 20], [pH5.0, 15% ethyleneglycol, 0.01% Tween 20], and [pH9.0, 15% ethylene glycol, 0.01% Tween20]. The buffers in which to make these solutions can readily bedetermined by one of skill in the art, and depend largely on the desiredpH of the final solution. Low pH solutions (<pH 5.5) can be made, forexample, in citrate buffer, glycine-HCl buffer, or in succinic acidbuffer. High pH solutions can be made, for example, in Tris-HCl,phosphate buffers, or sodium bicarbonate buffers. A number of conditionsmay be used to determine KD and off-rates for the purpose ofdetermining, for example, optimal pH and/or salt concentrations.

In one embodiment, a DBDpp specifically binds a target of interest witha K_(Off) ranging from 0.1 to 10⁻⁷ sec⁻¹, 10⁻² to 10⁻⁷ sec⁻¹, or0.5×10⁻² to 10⁻⁷ sec⁻¹. In a specific embodiment, a DBDpp (e.g., a DBDppfusion protein) binds a target of interest with an off rate (K_(Off)) ofless than 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹, or 10⁻³ sec⁻¹. In anadditional embodiment, a DBDpp, binds a target of interest with an offrate (K_(Off)) of less than 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or10⁻⁵ sec⁻¹, 10⁻⁵ sec⁻¹, 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹, or 10⁻⁷sec⁻¹.

In one embodiment, a DBDpp specifically binds a target of interest witha K_(On) ranging from 10³ to 10⁷ M⁻¹ sec-1, 10³ to 10⁶ M⁻¹ sec-1, or 10³to 10⁵ M⁻¹ sec-1. In other specific embodiments, a DBDpp (e.g., a DBDppfusion protein) binds the target of interest its target of interest withan on rate (K_(On)) of greater than 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴M⁻¹ sec⁻¹, or 5×10⁴ M⁻¹ sec⁻¹. In an additional embodiment, a DBDpp,binds a target of interest with a K_(On) of greater than 10⁵ M⁻¹ sec⁻¹,5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×10⁶ M⁻¹ sec⁻¹, or 10⁷ M⁻¹ sec⁻¹.

DBDpp Targets of Interest

The target of interest specifically bound by a DBDpp can be any moleculefor which it is desirable for a DBDpp to bind. For example, the targetsspecifically bound by DBDpp can be any target of purification,manufacturing, formulation, therapeutic, diagnostic, or prognosticrelevance or value. A number of exemplary targets are provided herein,by way of example, and are intended to be illustrative and not limiting.The target of interest can be naturally occurring or synthetic. Thetarget of interest can be an extracellular component or an intracellularcomponent, a soluble factor (e.g., an enzyme, hormone, cytokine, andgrowth factor, toxin, venom, pollutant, etc.), or a transmembraneprotein (e.g., a cell surface receptor). In one embodiment, the targetof interest specifically bound by a DBDpp is itself a DBDpp having adifferent sequence.

In one embodiment, a DBDpp fusion protein specifically binds a target ofinterest on the surface of a target cell. In a further embodiment, theDBDpp fusion protein specifically binds a cell surface receptor. In oneembodiment, a DBDpp fusion protein specifically binds a target ofinterest that is a member of a family selected from: a growth factorreceptor, a tyrosine kinase receptor, a TNF family receptor, aG-protein-coupled receptor, and a chemokine receptor. In someembodiments, the DBDpp fusion protein binds multiple members of the samefamily (e.g., the TNF receptors TRAILR1 and TRAILR2). In someembodiments, the DBDpp fusion protein binds members from differentfamilies. Thus, for example, in some embodiments, a DBDpp fusion proteincan bind to a growth factor receptor and a TNF receptor or aG-protein-coupled receptor and a chemokine receptor.

In one embodiment, a DBDpp specifically binds a serum protein or atherapeutic protein, such as an antibody or antibody fragment. In someembodiments, a target of interest bound by a DBDpp (e.g., a DBDpp fusionprotein) is a human protein. In one embodiment, a DBDpp (e.g., a DBDppfusion protein) binds a human protein target of interest and its monkey(e.g., cynomolgous monkey), mouse, rabbit, hamster and/or a rabbitortholog.

In one embodiment a DBDpp specifically binds a target of interest thatis a serum protein. In one embodiment, embodiment a DBDpp specificallybinds a serum protein selected from: serum albumin (e.g., human serumalbumin (HSA)), thyroxin-binding protein, transferrin, fibrinogen, andan immunoglobulin (e.g., IgG, IgE and IgM). Without being bound bytheory, the binding of a DBDpp to a carrier protein is believed toconfer upon the DBDpp (or a fusion thereof) an improved pharmacodynamicprofile that includes, but is not limited to, improved tumor targeting,tumor penetration, diffusion within the tumor, and enhanced therapeuticactivity compared to the DBDpp fusion protein in which the carrierprotein binding sequence is missing (see, e.g., WO 01/45746, thecontents of which are herein incorporated by reference in its entirety).

In one embodiment the target of interest specifically bound by a DBDppis a disease-related antigen. The antigen can be an antigencharacteristic of a cancer, and/or of a particular cell type (e.g., ahyperproliferative cell), and/or of a pathogen (e.g., a bacterial cell(e.g., tuberculosis, smallpox, and anthrax), a virus (e.g., HIV), aparasite (e.g., malaria and leishmaniosis), a fungal infection, a mold,a mycoplasm, a prion antigen, or an antigen associated with a disorderof the immune system.

In an additional embodiment, the target of interest bound by a DBDpp(e.g., a DBDpp fusion protein) is a bacterial antigen, a viral antigen,a fungal antigen, a mycoplasm antigen, a prion antigen, or a parasiteantigen (e.g., one infecting a mammal). In one embodiment, the target ofa DBDpp is anthrax, hepatitis b, rabies, Nipah virus, west Nile virus, ameningitis virus, or CMV. In an additional embodiment, a DBDppspecifically binds a pathogen.

In one embodiment, a DBDpp specifically binds a cancer target. Inanother embodiment, a DBDpp specifically binds a TSA or TAA. In someembodiments the DBDpp specifically binds a target selected from thegroup consisting of PTGER4, ITGA4, CD37, CD52, CD62L (L-selectin),CXCR4, CD69, EVI2B (CD361), SLC39A8, MICB, LRRC70, CLELC2B, HMHA1, LST1,and CMTM6 (CKLFSF6).

In one embodiment, a DBDpp specifically binds CD19 (B-CLL, B-ALL,leukemia, lymphoma, BNHL/CLL, ALL post-HCST, B lymphoid malignancies, Blineage malignancies), CD20 (mantle cell lymphoma/indolent B-NHL), PMSA(prostate cancer), CEA (breast cancer, colorectal cancer), Her2/neu(lung cancer, osteosarcoma, glioblastoma), kappa light chain (B-NHL andB-CLL).

In one embodiment, a DBDpp specifically binds a target selected from thegroup consisting of CD47, CTLA4, DR5, KIR, LAGS, OX40, PD-L1 and TIM3.

In one embodiment, a DBDpp specifically binds a target of interest isselected from the group consisting of: PDGFRA, PDGFRB, PDGFA, PDGFB,PDGFCC, PDGFC, PDGFD, VEGFR1, VEGFR2, VEGFR3, VEGFC, VEGFD, neuropilin 2(NRP2), betacellulin, PLGF, RET (rearranged during transfection), TIE1,TIE2 (TEK), CA125, CD3, CD4, CD7, CD10, CD13, CD19, CD22, CD25, CD30,CD32, CD32b, CD33, CD38, FRSF5 (CD40), CD44 (e.g., CD44v6), CD47, CD49e(integrin alpha 5), CD52, CD54 (ICAM), CD55, CD64, CD74, CD80, CD90,CD117 (cKit), CD133, CD200, (prominin 1), CD147, CD166, CD200, ESA, SHH,DHH, IHH, patched 1 (PTCH1), smoothened (SMO), WNT1, WNT2B, WNT3A, WNT4.WNT4A, WNT5A, WNT5B, WNT7B, WNT8A, WNT10A, WNT10B, WNT16B, LKP5, LRP5,LRP6, FZD1, FZD2, FZD4, FZD5, FZD6, FZD7, FZD8, Notch, Notch1, Notch3,Notch4, DLL4, Jagged, Jagged1, Jagged2, Jagged3, TNFSF1 (TNFb, LTa),TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFSF6 (Fas Ligand),TNFRSF6 (Fas, CD95), TNFRSF6B (DcR3), TNFSF4 (OX40 Ligand), TNFSF5 (CD40Ligand), TNFSF7 (CD27 Ligand, CD70), TNFRSF7 (CD27), TNFSF8 (CD30Ligand), TNFSF9 (41BB Ligand), TNFRSF8 (CD30), TNFSF11 (RANKL),TNFRSF10A (TRAILR1, DR4), TNFRSF10B (TRAILR2, DR5), TNFRSF4 (OX40),TNFRSF11A (RANK), TNFSF12 (TWEAK), TNFRSF12 (TWEAKR), TNFSF13 (APRIL),TNFSF13B (BLYS), TNFRSF13B (TACI), TNFRSF13C (BAFFR), TNFSF15 (TL1A),TNFRSF17 (BCMA), TNFRSF19L (KELT), TNFRSF19 (TROY), TNFRSF21 (DR6),TNFRSF25 (DR3), ANG1 (ANGPT1), ANG2 (ANGPT2), ANG3 (ANGPTL1), ANG4(ANGPT4), TIE2, IL1 alpha, IL1 beta, ILIRI, 1L1R2, IL2 IL2R, IL5, IL5R,IL6, IL6R, 1L8, 1L8R, IL10, IL10R, IL12, IL12R, IL13, IL13R, IL15,IL15R, IL18, IL18R, IL19, IL19R, IL21, IL21R, IL23, IL23R, mif, XAG1,XAG3, REGIV, FGF1, FGF2, FGF3, FGF4, FGFR1, FGFR2, FGFR3, ALK, ALK1,ALK7, ALCAM, Artemin, Axl, TGFb, TGFb2, TGFb3, TGFBR1, IGFIIR, BMP2,BMP5, BMP6, BMPRI, GDF3, GDF8, GDF9, N-cadherin, E-cadherin,VE-cadherin, EPCAM (EGP2), NCAM, LI CAM (GDI 71), ganglioside GM2,ganglioside GD2, calcitonin, PSGR, DCC, CDCP1, CXCR2, CXCR7, CCR3, CCR4,CCR5, CCR7, CCR10, CXCR4, CXCL1, CXCL5, CXCL6, CXCL8, CXCL12, CCL2,CCL3, CCL4, CCL5, CCL11, Claudin1, Claudin2, Claudin3, Claudin4, TMEFF2,neuregulin, MCSF, CSF, CSFR (fms), GCSF, GCSFR, BCAM, HPV, hCG, SR1F,PSA, FOLR2 (folate receptor beta), BRCA1, BRCA2, HLA-DR, ABCC3, ABCB5,HM 1.24, LFA1, LYNX, S100A8, S100A9, SCF, Von Willebrand factor, LewisY6 receptor, Lewis Y, CA G250 (CA9), CRYPTO, VLA5, CTLA4, HLA-DR, MUCl,MUC 8, mucin CanAg, ganglioside GD3, EGFL7, PDGFRa, IL21, IGF1, IGF2,HGF, PSMA, SLAMF7, carcinoembryonic antigen (CEA), FAP, integrin avb3,integrin α5β activin B1 alpha, leukotriene B4 receptor (LTB4R),neurotensin NT receptor (NTR), 5T4 oncofetal antigen, Tenascin C, MMP,MMP2, MMP7, MMP9, MMP12, MMP14, MMP26, cathepsin G, cathepsin H,cathepsin L, SULF1, SULF2, MET, UP A, MHCL MN (CA9), TAG-72, TM4SF1,Heparanase (HPSE), syndecan (SDC1), Ephrin B2, Ephrin B4, T neuropilin 1(NRP1), TEM1, mesothelin, TGFbeta 1, TGFBRII, FcRn, phosphatidlyserine,folate receptor alpha (FOLR1), and relaxin2. The above targets and thoseotherwise described herein are intended to be illustrative and notlimiting.

In one embodiment, a DBDpp (e.g., a DBDpp fusion protein) specificallybinds a target of interest selected from: VEGF, VEGFA, VEGFR1, VEGFR2,IGF1R, integrin, cMet, EGFR, ErbB2 (Her2), CD20, nerve growth factor(NGR), hepatocyte growth factor receptor, ErbB3 (Her3), ErbB4, prostatespecific membrane antigen.

In one embodiment, a target of interest specifically bound by a DBDpp(e.g., a DBDpp fusion protein) is an antigen associated with anautoimmune disorder, inflammatory or other disorder of the immune systemor is associated with regulating an immune response.

In one embodiment, a DBDpp specifically binds a target of interest thatis an immunoinhibitory target. In another embodiment, a DBDppspecifically binds an immunoinhibitory target, selected from: IL1, IL1b,IL1Ra, IL5, IL6, IL6R, CD26L, CD28, CD80, FcRn, or FcGamma RIIB Inanother embodiment, a DBDpp specifically binds an immunostimulatorytarget selected from: CD25, CD28, CTLA4, PD1, B7-H1 (PD-L1), B7-H4,IL10, TGFbeta, TNFSF4 (OX40 Ligand), TNFRSF4 (OX40), TNFSF5 (CD40Ligand), TNFRSF5 (CD40), TNFSF9 (41BB Ligand), TNFRSF9 (41BB, CD137),TNFSF14 (LIGHT, HVEM Ligand), TNFRSF14 (HVEM), TNFSF15 (TL1A), TNFRSF25(DR3), TNFSF18 (GITR Ligand), and TNFRSF18 (GITR).

In an additional embodiment, a DBDpp specifically binds a target ofinterest selected from: IL1Rb, IL2, IL3, IL4, IL7, IL11, IL15, IL16,IL17, IL17A, IL17F, IL18, IL19, IL25, IL32, IL33, interferon beta, SCF,BCA1/CXCL13, CXCL1, CXCL2, CXCL6, CXCL13, CXCL16, C3AR, C5AR, CXCR1,CXCR2, CCR1, CCR3, CCR7, CCR8, CCR9, CCR10, ChemR23, CCL3, CCL5, CCL11,CCL13, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL24, CCL25, CCL26,CCL27, MPL, GP130, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TREM1,TREM2, oncostatin M, lymphotoxin alpha (LTa), integrin beta 7 subunit,CD49a (integrin alpha 1), integrin a5b3, MIF, ESM1, WIF1, cathepsin B,cathepsin D, cathepsin K, cathepsin S, TNFSF2 (TNFa), TNFSF3 (LTb),TNFRSF3 (LTBR), TNFSF6 (Fas Ligand), TNFRSF6 (Fas, CD95), TNFRSF6B(DcR3), TNFSF8 (CD30 Ligand), TNFRSF8 (CD30), TNFSF11 (RANKL), TNFRSF11A(RANK), TNFRSF16 (NGFR), TNFRSF19L (RELT), TNFRSF19 (TROY), TNFRSF21(DR6), CD14, CD23 CD36, CD36L, CD39, CD52, CD91, CD137, CD153, CD164,CD200, CD200R, BTLA, B7-1 (CD80), B7-2 (CD86), B7h, B7-DC (PDL2), ICOS,ICOSL, MHC, CD, B7-H2, B7-H3, B7x, SLAM, KIM-1, SLAMF2, SLAMF3, SLAMF4,SLAMF5, SLAMF6, and SLAMF7, TNFSF1A (TNF-alpha), TNFRSF1A (TNFR1, p55,p60), TNFRSF1B (TNFR2), TNFSF7 (CD27 Ligand, CD70), TNFRSF7 (CD27),TNFSF13B (BLYS), TNFSF13 (APRIL), TNFRSF13B (TACI), TNFRSF13C (BAFFR),TNFRSF17 (BCMA), TNFSF12 (TWEAK), TNFRSF12 (TWEAKR), TNFRSF5 (CD40),IL1, IL1b, IL1R, IL2R, IL4-Ra, IL5, IL5R, IL6, IL6R, IL9, IL12, IL13,IL14, IL15, IL15R, IL17f, IL17R, IL17Rb, IL17RC, IL20, IL21, IL22RA,IL23, IL23R, IL31, TSLP, TSLPR, interferon alpha, interferon gamma,B7RP1, cKit, GMCSF, GMCSFR, CTLA4, CD2, CD3, CD4, CD11a, CD18, CD20,CD22, CD30, CD40, CD86, CXCR3, CXCR4, CCR2, CCR4, CCR5, CCR8, CCL2,CXCL10, P1GF, alpha4 integrin subunit, A4B7 integrin, C5, RhD, IgE, andRh.

In another embodiment, a DBDpp specifically binds a target of interestselected from: amyloid beta (Abeta), beta amyloid, complement factor D,PLP, ROBO4, ROBO, GDNF, NGF, LINGO, myostatin, oxidized LDL, gpIIB,gpIIIa, PCSK9, Factor VIII, integrin a2bB3, AOC3, mesothelin, DKK1,osteopontin, cathepsin K, TNFRSF19L (RELT), TNFRSF19 (TROY), andsclerostin.

In one embodiment, a DBDpp specifically binds a target of interestselected from the group consisting of: CD137, CD47, CTLA4, DR5, KIR,PD-L1, PD1 and TIM3.

In one embodiment a DBDpp specifically binds CD137. In a furtherembodiment, a DBDpp specifically binds CD137 and comprises an amino acidsequence selected from: (a)MGSWVEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEKLRQRAAFIRFRLQAYRHN (SEQ ID NO:12), (b) MGSWVEFANRLWAIDQRLFALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEHLRDQAAFIRHKLQAYRHN (SEQ ID NO:13), (c)MGSWYEFRHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEGLREAAAFIRAKLQAYRHN (SEQ ID NO:14), (d) MGSWYEFSMRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEALRAKAAYIRWKLQAYRHN (SEQ IDNO:15), (e) MGSWFEFNHRLWAINERLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVERLRSMAAFIRYKLQAYRHN (SEQ ID NO:16), (f) MGSWYEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEYLRETAA HIRTRLQAYRHN(SEQ ID NO:17), (g) MGSWYEFHYRLHAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEELRIKAAFIRDRLQAYRHN (SEQ ID NO:18), and (h)MGSWAEFKQRLAAIKTRLEALGGSEAELAAFLGEIWAFEMELAAYKGKGNPEVEALGREAAAIRMELQAYRHN (SEQ ID NO:19). Other DBDpp and polypeptides thatcompletely or partially (e.g., overlap with an epitope) bind to the sameepitope of CD137 as an above DBDpp are provided. Additionally, DBDpp andpolypeptides that completely or partially compete with an above DBDppfor binding to CD137 are also provided. Nucleic acids encoding the DBDppare also provided, as are vectors containing the nucleic acids and hostcells containing the nucleic acids and vectors.

In one embodiment a DBDpp specifically binds CD47. In a furtherembodiment, a DBDpp specifically binds CD47 and comprises an amino acidsequence selected from (a)MGSWYEFDLRLHAIYDRLVALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEILRDNAAYIRQMLQAYRHN (SEQ ID NO:20), (b) MGSWVEFANRLWAIDQRLFALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEHLRDQAAFIRHKLQAYRHN (SEQ ID NO:21), (c)MGSWTEFTYRLSAIEWRLWALGGSEAELAWFEQKIAFFEDFLQYYKGKGNPEVEALKHEAGAILNELMAYRHN (SEQ ID NO:22), (d) MGSWAEFDHRLHAIRERLHALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEILRGNAAYIRALLQAYRHN (SEQ IDNO:23), and (e) MGSWTEFVGRLAAIEFRLWALGGSEAELAWFEAHIAFFEDYLQWYKGKGNPEVEALREEAGAIMEELKAYRHN (SEQ ID NO:24). Other DBDpp andpolypeptides that completely or partially bind to the same epitope ofCD47 as an above DBDpp are provided. Additionally, DBDpp andpolypeptides that completely or partially compete with an above DBDppfor binding to CD47 are also provided. Nucleic acids encoding the DBDppare also provided, as are vectors containing the nucleic acids and hostcells containing the nucleic acids and vectors.

In one embodiment a DBDpp specifically binds CTLA4. In a furtherembodiment, a DBDpp specifically binds CTLA4 and comprises an amino acidsequence of MGSWHEFHDRLQAIHERLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVESLRIAAAHI RQVLQAYRHN(SEQ ID NO:25). Other DBDpp and polypeptides that completely orpartially bind to the same epitope of CTLA4 as the above DBDpp areprovided. Additionally, DBDpp and polypeptides that completely orpartially compete with the above DBDpp for binding to CTLA4 are alsoprovided. Nucleic acids encoding the DBDpp are also provided, as arevectors containing the nucleic acids and host cells containing thenucleic acids and vectors.

In one embodiment, a DBDpp specifically binds DR5. In a furtherembodiment, a DBDpp specifically binds DR5 and comprises an amino acidsequence selected from (a) MGSWNYFKDHLAWIKNSLEALGGSEAELAHFETAIASFERQLQEYKGKGNPEVEALRK EAAAIRDELQAYRHN(SEQ ID NO:26), (b) MGSWLYFKEHLAHIKAWLEALGGSEAELAHFELAIADFEYHLQEYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:27), (c)MGSWTEFTYRLSAIEWRLWALGGSEAELAWFEQKIAFFEDFLQYYKGKGNPEVEALKHEAGAILNELMAYRHN (SEQ ID NO:28), (d) MGSWFYFKQHLAWIKSYLEALGGSEAELAHFERAIAAFEQHLQMYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ IDNO:29), (e) MGSWHYFKDHLAEIKGLLEALGGSEAELAHFEMAIADFEHNLQYYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:30), (f) MGSWHYFKGHLAEIKNHLEALGGSEAELAHFERAIAAFERSLQWYKGKGNPEVEALRKEAA AIRDELQAYRHN(SEQ ID NO:31), (g) MGSWIYFKEHLAYIKKELEALGGSEAELAHFESAIAVFESTLQYYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:32), (h)MGSWTYFKEHLAEIKYMLEALGGSEAELAHFEVAIADFEKMLQYYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:33), and (i) MGSWWLFKDHLAEIKTALEALGGSEAELAHFEMAIAAFEKQLQYYKGKGNPEVEALRKEAAAIRDEL QAYRHN (SEQ IDNO:34). Other DBDpp and polypeptides that completely or partially bindto the same epitope of DR5 as an above DBDpp are provided. Additionally,DBDpp and polypeptides that completely or partially compete with anabove DBDpp for binding to DR5 are also provided. Nucleic acids encodingthe DBDpp are also provided, as are vectors containing the nucleic acidsand host cells containing the nucleic acids and vectors.

In one embodiment, a DBDpp specifically binds KIR. In a furtherembodiment, a DBDpp specifically binds KIR and comprises an amino acidsequence selected from (a) MGSWSEFYNRLDAIESRLLALGGSEAELALFEIQIARFEKVLQAYKGKGNPEVEALR GEARAIFAELYAYRHN(SEQ ID NO:35), (b) MGSWYEFYNRLYAIEIRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVERLRVRAAKIRVILQAYRHN (SEQ ID NO:36), and (c)MGSWLWFKIFLAEIKYFLEALGGSEAELAAFDFEIHAFHVELFAYKGKGNPEVEVLREVAAEIRWDLQAYRHN (SEQ ID NO:37). Other DBDpp and polypeptidesthat completely or partially bind to the same epitope of KIR as an aboveDBDpp are provided. Additionally, DBDpp and polypeptides that completelyor partially compete with an above DBDpp for binding to KIR are alsoprovided. Nucleic acids encoding the DBDpp are also provided, as arevectors containing the nucleic acids and host cells containing thenucleic acids and vectors.

In one embodiment, a DBDpp specifically binds PD-L1. In a furtherembodiment, a DBDpp specifically binds PD-L1 and comprises an amino acidsequence selected from (a)MGSWTEFQSRLDAIHSRLRALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVELLRDDAAFIRHFLQAYRHN (SEQ ID NO:38), (b) MGSWQEFDDRLNAIKARLQALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEDLRDDAAFIRRFLQAYRHN (SEQ ID NO:39), (c)MGSWYEFQNRLHAIHERLNALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVELLRDDAAFIRHFLQAYRHN (SEQ ID NO:40), (d) MGSWFEFQDRLTAINERLSALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVETLRSDAAFIRRFLQAYRHN (SEQ ID NO:41),(e) MGSWYEFESRLDAIHERLHALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVENLRGDAAFIRHFLQAYRHN (SEQ ID NO:42), (f) MGSWYEFNHRLDAISKRLNALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEELRGDAAFIRHFL QAYRHN (SEQ IDNO:43), and (g) MGSWFEFENRLHAIVHRLGALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVETLRADAAFIRHYLQAYRHN (SEQ ID NO:44). Other DBDppand polypeptides that completely or partially bind to the same epitopeof PD-L1 as an above DBDpp are provided. Additionally, DBDpp andpolypeptides that completely or partially compete with a DBDpp forbinding to PD-L1 are also provided. Nucleic acids encoding the DBDpp arealso provided, as are vectors containing the nucleic acids and hostcells containing the nucleic acids and vectors.

In one embodiment, a DBDpp specifically binds PD1. In a furtherembodiment, a DBDpp specifically binds PD1 and comprises an amino acidsequence selected from (a)MGSWTIFKEWLAFIKTDLEALGGSEAELAFFEGWIASFEMELQKYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:46), (b) MGSWVMFKWLLADIKSHLEALGGSEAELAFFEGFIAAFETHLQVYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:47), and(c) MGSWYAFKDYLADIKGWLEALGGSEAELAFFEIFIARFELELQAYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:48). Other DBDpp andpolypeptides that completely or partially bind to the same epitope ofPD1 as an above DBDpp are provided. Additionally, DBDpp and polypeptidesthat completely or partially compete with an above DBDpp for binding toPD1 are also provided. Nucleic acids encoding the DBDpp are alsoprovided, as are vectors containing the nucleic acids and host cellscontaining the nucleic acids and vectors.

In one embodiment, a DBDpp specifically binds TIM3. In a furtherembodiment, a DBDpp specifically binds TIM3 and comprises an amino acidsequence of MGSWHEFHDRLQAIHERLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVESLRIAAAHIRQV LQAYRHN (SEQID NO:45). Other DBDpp and polypeptides that completely or partiallybind to the same epitope of TIM3 as the above DBDpp are provided.Additionally, DBDpp and polypeptides that completely or partiallycompete with the DBDpp for binding to TIM3 are also provided. Nucleicacids encoding the DBDpp are also provided, as are vectors containingthe nucleic acids and host cells containing the nucleic acids andvectors.

In another embodiment, the DBDpp binds a peptide tag present on a targetof interest. Such peptide tags provide a useful means by which topurify, detect and/or attach targets of interest containing the peptidetags. In one embodiment, a DBDpp specifically binds a peptide tagselected from the group: a hexahistidyl (His6) tag, a myc tag or a FLAGtag. Other peptide tags are described herein or otherwise known in theart.

In another embodiment, the target to which DBDpp binds is the subject ofpurification from a mixture of contaminants. In one embodiment thetarget may be a natural or recombinantly expressed protein that requiresselective isolation from a cell lysate or cell culture supernatant.

DBDpp Fusion Proteins

A “fusion polypeptide,” “fusion protein,” “chimeric polypeptide,”“chimeric protein,” “chimeric antigen” is a polypeptide comprised of atleast two polypeptides and optionally a linker to operatively link thetwo polypeptides into one continuous polypeptide produced, e.g., byrecombinant processes. The two polypeptides may be operably attacheddirectly or indirectly.

A “DBDpp fusion protein” comprises at least one DBDpp that specificallybinds a target of interest. In one embodiment, the DBDpp fusion proteinscomprise more than one DBDpp, wherein the two or more DBDpp have thesame or different specificities. In additional embodiments, the DBDppfusion protein is comprised of a tandem repeat of the same or differentDBDpp that allow a DBDpp fusion protein to bind multiple targets and/orrepeating epitopes or different epitopes on the same target. Inadditional embodiments, a DBDpp fusion protein comprises a DBDpp and apolypeptide sequence containing an additional domain. In someembodiments, the DBDpp fusion protein comprises a DBDpp and a memberselected from: an antibody, an antibody fragment (e.g., an antigenbinding domain or portion thereof (e.g., an ScFv), an effector domain orportion thereof, an FcRn binding domain or portion thereof, and an Fc ora portion thereof), a serum protein (e.g., albumin or a portionthereof), a cytokine, a growth factor, a hormone, an imaging agent, alabeling agent, and a peptide tag. In some embodiments, the DBDpp fusionprotein comprises an Fc domain of an immunoglobulin (e.g., a human Fcdomain) or a portion thereof. In further embodiments, the Fc domain is avariant human Fc domain.

The DBDpp provided herein include DBDpp fusion proteins. A DBDpp and anypolypeptide of interest can be operably linked to form a DBDpp fusionprotein. Thus, in some embodiments, the DBDpp is incorporated into alarger, multi-domain molecular complex (e.g., a monomeric or multimericDBDpp fusion protein) and in so doing, imparts the functional attributesof the incorporated DBDpp to the resultant fusion protein. In someembodiments, DBDpp fusion proteins comprise a DBDpp and a polypeptidesequence from an antibody, an antibody fragment, a serum protein (e.g.,human serum albumin) or serum protein fragment, or a cell surfacereceptor, an alpha chain of a T cell receptor (TCR), a beta chain of a Tcell receptor, cytokine, growth factor, hormone, or enzyme, or fragmentthereof. Incorporation of DBD into multidomain and/or multifunctionalcomplexes can routinely be achieved by way of recombinant fusion toanother polypeptide, binding to another chemical moiety, and covalentchemical linkage to another polypeptide (or other desirable chemicalcompound) using techniques known in the art. DBDpp fusion proteins canadditionally contain other optional components such as linkers and othercomponents described herein.

DBDpp Multimers

In some embodiments, the DBDpp fusion protein contains one DBDpp. Insome embodiments, the DBDpp fusion protein comprises at least 2, 3, 4,or 5, or more than 5 DBDpp. In some embodiments, the DBDpp fusionprotein contains 1-3, 1-4, 1-5, or more than 5 different DBDpp. In someembodiments, the DBDpp fusion protein contains at least 2, 3, 4, or 5,or more than 5 different DBDpp. Thus, a DBDpp fusion protein can be amonomeric DBDpp (i.e., containing one DBDpp) or multimeric DBDpp (i.e.,containing more than one DBDpp in tandem optionally operably connectedby a linker). Non-limiting embodiments of such multimeric DBDpp areshown in FIG. 5A. In several embodiments, the use of multimeric DBDppprovides enhanced (e.g., synergistic) target binding. In additionalembodiments, multimeric DBDpp allows targeting of more than one targetusing a single DBDpp construct (e.g., bi-, tri-specific, etc.).

The multimeric DBDpp fusion protein can be a DBDpp homo-multimeric(i.e., containing more than one of the same DBDpp in tandem optionallyconnected by linker(s) (e.g., homodimers, homotrimers, homotetramersetc.) or DBDpp hetero-multimeric (i.e., containing two or more DBDpp inwhich there are at least two different DBDpp protein. The number ofmonomeric DBDpp included within a multimeric composition may vary,depending on the embodiment, and may be defined, at least in part, bythe expression system in which the DBDpp is produced. In severalembodiments, however, the fusion proteins may comprises multimers ofabout 5 to about 10 DBDpp subunits, about 10 to about 15 subunits, about15 to about 20 subunits, about 20 to about 25 subunits, or about 25 toabout 30 subunits (including numbers in between those listed as well asendpoints). Moreover, multiple tandem components of a DBDpp fusionprotein can contain the same or different DBDpp. In some DBDpp fusions,the DBDpp are present as a monomer, or in homomultimers or heteromerssuch as, homodimers or heterodimers, homotrimers or heterotrimers,homotetramers or heterotetramers.

In one embodiment, two or more DBDpp are operably fused to form a DBDppfusion protein. In one embodiment, the fusion partner of a DBDpp is anidentical DBDpp. The linkage of two or more identical DBDpp results in amultivalent molecule that provides distinct advantages (e.g., increasedbinding avidity, target clustering and receptor activation) overmonomeric compositions. In another embodiment the fusion partner of aDBDpp is a non-identical DBDpp. The linkage of two or more non-identicalDBDpp results in a multivalent and multi-specific molecule that has thepotential to bind more than one target antigen, either independently orsimultaneously.

A DBDpp fusion protein can be “monospecific” or “multi-specific.” ADBDpp fusion protein that is “multi-specific” (e.g., bispecific,trispecific or of greater multi-specificity) recognizes and binds to twoor more different epitopes present on one or more different molecules(e.g., proteins, solid support structures, etc.).

In one embodiment, a multi-specific DBDpp fusion protein contains atleast two DBDpp that bind to at least two different epitopes on a singletarget of interest. In additional embodiments, a multi-specific DBDppfusion protein comprises at least one DBDpp that specifically binds oneepitope on a target of interest and at least one other domain orsequence conferring function (e.g., an antibody fragment or domain suchas an scFv) that specifically binds to a different epitope on the sametarget of interest. In one embodiment, a multi-specific DBDpp fusionprotein comprises at least one DBDpp that specifically binds to anepitope on a target of interest and at least one domain or sequenceconferring function e.g., an antibody fragment or domain (e.g., scFv),that specifically binds to an epitope on a different target of interest.In other embodiments, a DBDpp fusion protein comprises at least oneDBDpp and at least one other DBDpp or domain sequence conferringfunction, e.g., an antibody fragment or domain, that specifically bindsto a solid support.

In a further embodiment, the multimeric DBDpp fusion comprising 2 ormore DBDpp are in turn fused with other heterologous proteins (or theirsubdomains) and in so doing, impart the multivalent and multi-specificproperties to the fusion partner. Examples of fusion partners of a DBDppincludes but is not limited to, antibodies, antibody subdomains (e.g.,scFv or Fc domains), serum albumin, serum albumin subdomains, cellsurface receptors, an alpha chain of a T cell receptor (TCR), a betachain of a T cell receptor, cell surface receptor subdomains, peptides,peptide tags (e.g., FLAG or myc), fibronectin type III repeats,z-domains, elastin-like polypeptides. The number and location of DBDppand their respective positions within the fusion protein can vary. Forexample, DBDpp(s) can be located at one or all termini of a fusionpartner and/or interspersed within heterologous subunits within theDBDpp fusion partner.

In one embodiment, the DBDpp fusion is bispecific and specifically bindsto two different targets expressed on the surface of two different celltypes. In one embodiment the bispecific DBDpp fusion proteinspecifically binds to a cancer cell target and an immune effector celltarget. In one embodiment the bispecific DBDpp fusion proteinspecifically binds a target expressed on a cancer cell (e.g. CD19) and atarget expressed on the surface of a T lymphocyte (e.g., CD3).

DBDpp as Fusions to Antibodies and Antibody Fragments

In one embodiment, a DBDpp fusion protein comprises a whole antibody oran antibody fragment or domain (e.g., an IgG1 antibody, IgG3 antibody,antibody variable region, CDR3, ScFv, Fc, FcRn binding domain, and otherantibody domains). DBDpp and DBDpp fusion proteins can be operablylinked to one another and/or to one or more termini of an antibody,antibody chain, antibody fragment or antibody domain.

The antibody component of a DBDpp fusion protein can be any suitablewhole immunoglobulin or antibody fragment (e.g., an antigen bindingdomain and/or effector domain) or a fragment thereof. In one embodiment,the DBDpp-antibody fusion protein retains the structural and functionalproperties of a traditional monoclonal antibody. Thus, in someembodiments, the DBDpp-antibody fusion protein retains the epitopebinding properties, but advantageously also incorporate, via the DBDppfusion, one or more additional target-binding specificities. Antibodiesthat can be used in the DBDpp fusions include, but are not limited to,monoclonal, multi-specific, human, humanized, primatized, and chimericantibodies. Immunoglobulin or antibody molecules provided herein can beof any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.In specific embodiments, the antibodies are Fc optimized antibodies.Antibodies can be from or derived from any animal origin including birdsand mammals or generated synthetically. The antibody component of theDBDpp-antibody fusion protein can be naturally derived or the result ofrecombinant engineering (e.g., phage display, xenomouse, and synthetic).In certain embodiments, the antibody component of the antibody-DBDppfusion enhances half-life, and increase or decrease antibody dependentcellular cytotoxicity (ADCC), and/or complement dependent cytotoxicity(CDC) activity. In some embodiments, the antibodies are human, murine,donkey, rabbit, goat, guinea pig, camel, llama, horse, or chickenantibodies. In specific embodiments, the antibodies are human.

In one embodiment, a DBDpp is operably linked to an antibody fragment orsubdomain (e.g., ScFv, diabody, EP 404,097; WO 93/111161; WO2014/028776; and Holliger et al., PNAS 90:6444-6448 (1993), each ofwhich are herein incorporated by reference in its entirety). Theantibody fragment or subdomain can be any fragment or domain of anantibody. See for example, WO 04/058820, WO 99/42077 and WO 05/017148,each of which is herein incorporated by reference in its entirety. Forexample, a DBDpp fusion protein can contain an antibody effector domainor derivative of an antibody effector domain that confers one or moreeffector functions to the DBDpp and/or confers upon the DBDpp fusionprotein the ability to bind to one or more Fc receptors. In someembodiments, a DBDpp-antibody fusion protein contains an antigen-bindingfragment of an antibody or a fragment thereof. In additionalembodiments, a DBDpp-antibody fusion protein contains an immunoglobulineffector domain that comprises one or more CH2 and or CH3 domains of anantibody having effector function provided by the CH2 and CH3 domains.Other sequences in the DBDpp fusion that provide an effector functionand that are encompassed by the invention will be clear to those skilledin the art and can routinely be chosen and designed into a DBDpp fusionprotein encompassed herein on the basis of the desired effectorfunction(s).

In one embodiment, the antibody component of an antibody-DBDpp fusionprovided herein has been modified to increase antibody dependentcellular cytotoxicity (ADCC) (see, e.g., Bruhns et al., Blood113:3716-3725 (2009); Shields et al., J. Biol. Chem. 276:6591-6604(2001); Lazar et al., PNAS 103:4005-4010 (2006); Stavenhagen et al.,Cancer Res., 67:8882-8890 (2007); Horton et al., Cancer Res.68:8049-8057 (2008); Zalevsky et al., Blood 113:3735-3743 (2009);Bruckheimer, Neoplasia 11:509-517 (2009); WO2006/0201 14; Strohl, Curr.Op. Biotechnol. 20:685-691 (2009); and WO2004/074455, each of which isherein incorporated by reference in its entirety). Examples of Fcsequence engineering modifications contained in the antibody componentof the DBDpp-antibody fusion proteins that increases ADCC include one ormore modifications corresponding to: IgG1-S298A, E333A, K334A;IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q;IgG1-D280H, K290S with or without S298D or V; IgG1-F243L, R292P, Y300L;IgG1-F243L, R292P, Y300L, P396L; and IgG1-F243L, R292P, Y300L, V305I,P396L; wherein the numbering of the residues in the Fc region is that ofthe EU index of Kabat et al. (Kabat et al., Sequences of proteins ofImmunological Interest, 1991 Fifth edition).

In one embodiment, the DBDpp fusion contains a whole antibody or anantibody fragment that is an antigen-binding fragment. In a furtherembodiment, the antibody or antibody fragment binds a disease-relatedantigen. In one embodiment the DBDpp fusion protein comprises anantibody or an antibody fragment that specifically binds a cancerantigen. In another embodiment, the DBDpp fusion protein comprises anantibody or an antibody fragment that specifically binds a particularpathogen (e.g., a bacterial cell (e.g., tuberculosis, smallpox,anthrax)), a virus (e.g., HIV), a parasite (e.g., malaria,leishmaniosis), a fungal infection, a mold, a mycoplasm, a prionantigen, In another embodiment, the DBDpp fusion protein comprises anantibody or an antibody fragment that specifically binds a particularpathogen (e.g., a bacterial cell (e.g., tuberculosis, smallpox,anthrax)), a virus (e.g., HIV), a parasite (e.g., malaria,leishmaniosis), a fungal infection, a mold, a mycoplasm, or a prionantigen. In another embodiment, the DBDpp fusion protein comprises anantibody or an antibody fragment that specifically binds an antigenassociated with a disease or disorder of the immune system.

In preferred embodiments, the DBDpp fusion protein containing anantibody fragment or domain retains activities of the parent antibody.Thus, in certain embodiments, the DBDpp fusion protein containing anantibody fragment or domain is capable of inducing complement dependentcytotoxicity. In certain embodiments, the DBDpp fusion proteincontaining an antibody fragment or domain is capable of inducingantibody dependent cell mediated cytotoxicity (ADCC).

Accordingly, in some embodiments, the DBDpp fusion protein comprises anantibody fragment that confers upon the DBDpp fusion protein abiological or biochemical characteristic of an immunoglobulin. In someembodiments, the antibody fragment confers a characteristic selectedfrom: the ability to non-covalently dimerize, the ability to localize atthe site of a tumor, and an increased serum half-life when compared tothe DBDpp fusion protein in which said one or more DBDpp have beendeleted. In certain embodiments, the DBDpp fusion protein is at least asstable as the corresponding antibody without the attached DBDpp. Incertain embodiments, the DBDpp fusion protein is more stable than thecorresponding antibody without the attached DBDpp. DBDpp fusion proteinstability can be measured using established methods, including, forexample, ELISA techniques. In some embodiments, the DBDpp fusion proteinis stable in whole blood (in vivo or ex vivo) at 37° C. for at leastabout 10 hours, at least about 15 hours, at least about 20 hours, atleast about 24 hours, at least about 25 hours, at least about 30 hours,at least about 35 hours, at least about 40 hours, at least about 45hours, at least about 48 hours, at least about 50 hours, at least about55 hours, at least about 60 hours, at least about 65 hours, at leastabout 70 hours, at least about 72 hours, at least about 75 hours, atleast about 80 hours, at least about 85 hours, at least about 90 hours,at least about 95 hours, or at least about 100 hours (including any timebetween those listed). In one embodiment, a DBDpp fusion contains animmunoglobulin effector domain or half-life influencing domain thatcorresponds to an immunoglobulin domain or fragment in which at least afraction of one or more of the constant region domains has been alteredso as to provide desired biochemical characteristics such as reduced orincreased effector functions, the ability to non-covalently dimerize,increased ability to localize at the site of a tumor, reduced serumhalf-life, or increased serum half-life when compared with animmunoglobulin fragment having the corresponding unalteredimmunoglobulin sequence. These alterations of the constant regiondomains can be amino acid substitutions, insertions, or deletions.

In one embodiment, a DBDpp fusion protein comprises an amino acidsequence of an immunoglobulin effector domain or a derivative of animmunoglobulin effector domain that confers antibody dependent cellularcytotoxicity (ADCC) to the DBDpp fusion protein. In additionalembodiments, a DBDpp fusion protein comprises a sequence of animmunoglobulin effector domain that has been modified to increase ADCC(see, e.g., Bruhns, Blood 113:3716-3725 (2009); Shields, J. Biol. Chem.276:6591-6604 (2001); Lazar, PNAS 103:4005-4010 (2006); Stavenhagen,Cancer Res. 67:8882-8890 (2007); Horton, Cancer Res. 68:8049-8057(2008); Zalevsky, Blood 113:3735-3743 (2009); Bruckheimer, Neoplasia11:509-517 (2009); WO 06/020114; Strohl, Curr. Op. Biotechnol.20:685-691 (2009); and WO 04/074455, the contents of each of which isherein incorporated by reference in its entirety). Examples ofimmunoglobulin fragment engineering modifications contained in an aminoacid sequence in a DBDpp fusion protein that increases ADCC includeimmunoglobulin effector domain sequences having one or moremodifications corresponding to: IgG1-S298A, E333A, K334A; IgG1-S239D,I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H,K290S with or without S298D or V; IgG1-F243L, R292P, Y300L; IgG1-F243L,R292P, Y300L, P396L; and IgG1-F243L, R292P, Y300L, V305I, P396L; whereinthe numbering of the residues in the Fc region is that of the EU indexof Kabat et al. (Kabat et al., Sequences of proteins of ImmunologicalInterest, 1991 Fifth edition, herein incorporated by reference).

In other embodiments, a DBDpp fusion protein comprises a sequence of animmunoglobulin effector domain that has been modified to decrease ADCC(see, e.g., Idusogie et al., J. Immunol. 166:2571-2575 (2001); Sazinskyet al., PNAS 105:20167-20172 (2008); Davis et al., J. Rheumatol.34:2204-2210 (2007); Bolt et al., Eur. J. Immunol. 23:403-411 (1993);Alegre et al., Transplantation 57:1537-1543 (1994); Xu et al., CellImmunol. 200:16-26 (2000); Cole et al., Transplantation 68:563-571(1999); Hutchins et al., PNAS 92:11980-11984 (1995); Reddy et al., J.Immunol. 164:1925-1933 (2000); WO 97/11971; WO 07/106585; US2007/0148167A1; McEarchern et al., Blood 109:1185-1192 (2007); Strohl,Curr. Op. Biotechnol. 20:685-691 (2009); and Kumagai et al., J. Clin.Pharmacol. 47:1489-1497 (2007), the contents of each of which is hereinincorporated by reference in its entirety). Examples of immunoglobulinfragment sequence engineering modifications contained in an amino acidsequence in a DBDpp fusion protein that decreases ADCC includeimmunoglobulin effector domain sequences having one or moremodifications corresponding to: IgG1-K326W, E333S; IgG2-E333S;IgG1-N297A; IgG1-L234A, L235A; IgG2-V234A, G237A; IgG4-L235A, G237A,E318A; IgG4-S228P, L236E; IgG2-118-260; IgG4-261-447; IgG2-H268Q, V309L,A330S, A331S; IgG1-C220S, C226S, C229S, P238S; IgG1-C226S, C229S, E233P,L234V, L235A; or IgG1-L234F, L235E, P331S; wherein the numbering of theresidues is that of the EU index of Kabat et al. (Kabat et al.,Sequences of Proteins of Immunological Interest, 1991 Fifth edition,herein incorporated by reference).

In additional embodiments, a DBDpp fusion protein comprises an aminoacid sequence of an immunoglobulin effector domain, or a derivative ofan immunoglobulin effector domain, that confers antibody-dependent cellphagocytosis (ADCP) to the DBDpp fusion protein. In additionalembodiments, a DBDpp fusion protein comprises a sequence of animmunoglobulin effector domain that has been modified to increaseantibody-dependent cell phagocytosis (ADCP); (see, e.g., Shields et al.,J. Biol. Chem. 276:6591-6604 (2001); Lazar et al., PNAS 103:4005-4010(2006); Stavenhagen et al., Cancer Res., 67:8882-8890 (2007); Richardset al., Mol. Cancer Ther. 7:2517-2527 (2008); Horton et al., Cancer Res.68:8049-8057 (2008), Zalevsky et al., Blood 113:3735-3743 (2009);Bruckheimer et al., Neoplasia 11:509-517 (2009); WO 06/020114; Strohl,Curr. Op. Biotechnol. 20:685-691 (2009); and WO 04/074455, the contentsof each of which is herein incorporated by reference in its entirety).Examples of immunoglobulin fragment engineering modifications containedin an amino acid sequence in a DBDpp fusion protein that increases ADCPinclude immunoglobulin effector domain sequences having one or moremodifications corresponding to: IgG1-S298A, E333A, K334A; IgG1-S239D,I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H,K290S with or without S298D or V; IgG1-F243L, R292P, Y300L; IgG1-F243L,R292P, Y300L, P396L; IgG1-F243L, R292P, Y300L, V305I, P396L; andIgG1-G236A, S239D, I332E; wherein the numbering of the residues is thatof the EU index of Kabat et al. (Kabat et al., Sequences of proteins ofImmunological Interest, 1991 Fifth edition, herein incorporated byreference).

In other embodiments, a DBDpp fusion protein comprises a sequence of animmunoglobulin effector domain that has been modified to decrease ADCP(see, e.g., Sazinsky et al., PNAS 105:20167-20172 (2008); Davis et al.,J. Rheumatol. 34:2204-2210 (2007); Bolt et al., Eur. J. Immunol.23:403-411 (1993); Alegre et al., Transplantation 57:1537-1543 (1994);Xu et al., Cell Immunol. 200:16-20 (2000); Cole et al., Transplantation68:563-571 (1999); Hutchins et al., PNAS 92:11980-11984 (1995); Reddy etal., J. Immunol. 164:1925-1933 (2000); WO 97/11971; WO 07/106585; US2007/0148167A1; McEarchern et al., Blood 109:1185-1192 (2007); Strohl,Curr. Op. Biotechnol. 20:685-691 (2009); and Kumagai et al., J. Clin.Pharmacol. 47:1489-1497 (2007), the contents of each of which is hereinincorporated by reference in its entirety). By way of example, DBDppfusion proteins can contain an antibody fragment or domain that containsone or more of the following modifications that decrease ADCC:IgG1-N297A; IgG1-L234A, L235A; IgG2-V234A, G237A; IgG4-L235A, G237A,E318A; IgG4-S228P, L236E; IgG2 EU sequence 118-260; IgG4-EU sequence261-447; IgG2-H268Q, V309L, A330S, A331S; IgG1-C220S, C226S, C229S,P238S; IgG1-C226S, C229S, E233P, L234V, L235A; and IgG1-L234F, L235E,P331S; wherein the numbering of the residues is that of the EU index ofKabat et al. (Kabat et al., Sequences of proteins of ImmunologicalInterest, 1991 Fifth edition, herein incorporated by reference).

In additional embodiments, a DBDpp fusion protein comprises an aminoacid sequence of an immunoglobulin effector domain, or a derivative ofan immunoglobulin effector domain, that confers complement-dependentcytotoxicity (CDC) to the DBDpp fusion protein. In additionalembodiments, a DBDpp fusion protein comprises a sequence of animmunoglobulin effector domain that has been modified to increasecomplement-dependent cytotoxicity (CDC) (see, e.g., Idusogie et al., J.Immunol. 166:2571-2575 (2001); Strohl, Curr. Op. Biotechnol. 20:685-691(2009); and Natsume et al., Cancer Res. 68:3863-3872 (2008), thecontents of each of which is herein incorporated by reference in itsentirety). By way of example, DBDpp fusion proteins can contain anantibody fragment or domain that contains one or more of the followingmodifications that increase CDC: IgG1-K326A, E333A; IgG1-K326W, E333S,IgG2-E333S; wherein the numbering of the residues is that of the EUindex of Kabat et al. (Kabat et al., Sequences of proteins ofImmunological Interest, 1991 Fifth edition, herein incorporated byreference).

In additional embodiments, a DBDpp fusion protein comprises an aminoacid sequence of an immunoglobulin effector domain, or a derivative ofan immunoglobulin effector domain, that confers the ability to bindFcgammaRIIb receptor to the DBDpp fusion. In additional embodiments, aDBDpp fusion protein comprises a sequence of an immunoglobulin effectordomain that has been modified to increase inhibitory binding toFcgammaRIIb receptor (see, e.g., Chu et al., Mol. Immunol. 45:3926-3933(2008)). An example of an immunoglobulin fragment engineeringmodification contained in an amino acid sequence in a DBDpp fusionprotein that increases binding to inhibitory FcgammaRIIb receptor isIgG1-S267E, L328F.

In other embodiments, a DBDpp fusion protein comprises a sequence of animmunoglobulin effector domain that has been modified to decrease CDC(see, e.g., WO 97/11971; WO 07/106585; US 2007/0148167A1; McEarchern etal., Blood 109:1185-1192 (2007); Hayden-Ledbetter et al., Clin. Cancer15:2739-2746 (2009); Lazar et al., PNAS 103:4005-4010 (2006);Bruckheimer et al., Neoplasia 11:509-517 (2009); Strohl, Curr. Op.Biotechnol. 20:685-691 (2009); and Sazinsky et al., PNAS 105:20167-20172(2008); the contents of each of which is herein incorporated byreference in its entirety). By way of example, DBDpp fusion proteins cancontain an antibody fragment or domain that contains one or more of thefollowing modifications that decrease CDC: IgG1-S239D, A330L, 1332E;IgG2-118-260; IgG4-261-447; IgG2-H268Q, V309L, A330S, A331S; IgG1-C226S,C229S, E233P, L234V, L235A; IgG1-L234F, L235E, P331S; and IgG1-C226S,P230S; wherein the numbering of the residues is that of the EU index ofKabat et al. (Kabat et al., Sequences of proteins of ImmunologicalInterest, 1991 Fifth edition, herein incorporated by reference).

The half-life of an IgG is mediated by its pH-dependent binding to theneonatal receptor FcRn. In certain embodiments a DBDpp fusion proteincomprises an amino acid sequence of an immunoglobulin effector domain,or a derivative of an immunoglobulin effector domain, that confers theability to bind neonatal receptor FcRn to the to the DBDpp fusion. Incertain embodiments a DBDpp fusion protein comprises a sequence of animmunoglobulin FcRn binding domain that has been modified to enhancebinding to FcRn (see, e.g., Petkova et al., Int. Immunol. 18:1759-1769(2006); Dall'Acqua et al., J. Immunol.169:5171-5180 (2002); Oganesyan etal., Mol. Immunol. 46:1750-1755 (2009); Dall'Acqua et al., J. Biol.Chem. 281:23514-23524 (2006), Hinton et al., J. Immunol. 176:346-356(2006); Datta-Mannan et al., Drug Metab. Dispos. 35:86-94 (2007);Datta-Mannan et al., J. Biol. Chem. 282:1709-1717 (2007); WO 06/130834;Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Yeung et al., J.Immunol. 182:7663-7671 (2009) the contents of each of which is hereinincorporated by reference in its entirety).

In additional embodiments, a DBDpp fusion protein comprises a sequenceof an immunoglobulin effector domain that has been modified to have aselective affinity for FcRn at pH 6.0, but not pH 7.4. By way ofexample, DBDpp fusion proteins can contain an antibody fragment ordomain that contains one or more of the following modifications thatincrease half-life: IgG1-M252Y, S254T, T256E; IgG1-T250Q, M428L;IgG1-H433K, N434Y; IgG1-N434A; and IgG1-T307A, E380A, N434A; wherein thenumbering of the residues is that of the EU index of Kabat et al. (Kabatet al., Sequences of Proteins of Immunological Interest, 1991 Fifthedition, herein incorporated by reference).

In other embodiments a DBDpp fusion protein comprises a sequence of animmunoglobulin effector domain that has been modified to decreasebinding to FcRn (see, e.g., Petkova et al., Int. Immunol. 18:1759-1769(2006); Datta-Mannan et al., Drug Metab. Dispos. 35:86-94 (2007);Datta-Mannan et al., J. Biol. Chem. 282:1709-1717 (2007); Strohl, Curr.Op. Biotechnol. 20:685-691 (2009); and Vaccaro et al., Nat. Biotechnol.23:1283-1288 (2005), the contents of each of which is hereinincorporated by reference in its entirety). By way of example, DBDppfusion proteins can contain an antibody fragment or domain that containsone or more of the following modifications that decrease half-life:IgG1-M252Y, S254T, T256E; H433K, N434F, 436H; IgG1-I253A; andIgG1-P2571, N434H and D376V, N434H; wherein the numbering of theresidues is that of the EU index of Kabat et al. (Kabat et al.,Sequences of proteins of Immunological Interest, 1991 Fifth edition,herein incorporated by reference).

According to another embodiment, DBDpp fusion protein comprises an aminoacid sequence corresponding to a immunoglobulin effector domain that hasbeen modified to contain at least one substitution in its sequencecorresponding to the Fc region (e.g., FC gamma) position selected fromthe group consisting of: 238, 239, 246, 248, 249, 252, 254, 255, 256,258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289,290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315,320, 322, 324, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338,340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434,435, 437, 438 and 439, wherein the numbering of the residues in the Fcregion is according to the EU numbering system; of Kabat et al. (Kabatet al., Sequences of proteins of Immunological Interest, 1991 Fifthedition, herein incorporated by reference). In a specific embodiment,the DBDpp fusion protein comprises a sequence of an immunoglobulineffector domain derivative wherein at least one residue corresponding toposition 434 is a residue selected from the group consisting of: A, W,Y, F and H. According to another embodiment, the DBDpp fusion proteincomprises a sequence of an immunoglobulin effector fragment derivativehaving the following respective substitutions S298A/E333A/K334A. In anadditional embodiment, the DBDpp fusion protein comprises animmunoglobulin effector domain derivative having a substitutioncorresponding to K322A. In another embodiment, the DBDpp fusion proteincomprises a sequence of an immunoglobulin effector domain derivativehaving one or any combination of the following substitutions K246H,H268D, E283L, S324G, S239D and 1332E. According to yet anotherembodiment, a DBDpp fusion protein comprises a sequence of animmunoglobulin effector domain derivative having substitutionscorresponding to D265A/N297A.

In certain embodiments, a DBDpp fusion protein comprises a sequence ofan immunoglobulin effector domain that has been glycoengineered ormutated to increase effector function using techniques known in the art.For example, the inactivation (through point mutations or other means)of a constant region domain sequence contained in a DBDpp may reduce Fcreceptor binding of the circulating DBDpp fusion protein therebyincreasing tumor localization. In other cases it may be that constantregion modifications consistent with certain embodiments of the instantinvention moderate complement binding and thus reduce the serumhalf-life and nonspecific association of a conjugated cytotoxin. Yetother modifications of the constant region may be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. The resulting physiological profile, bioavailability andother biochemical effects of the modifications, such as tumorlocalization, biodistribution and serum half-life, can easily bemeasured and quantified using well know immunological techniques withoutundue experimentation.

In certain embodiments an immune effector cell comprises a cell surfacereceptor for an immunoglobulin or other peptide binding molecule, suchas a receptor for an immunoglobulin constant region and including theclass of receptors commonly referred to as “Fc receptors” (“FcR”s). Anumber of FcRs have been structurally and/or functionally characterizedand are known in the art, including FcR having specific abilities tointeract with a restricted subset of immunoglobulin heavy chainisotypes, or that interact with Fc domains with varying affinities,and/or which may be expressed on restricted subsets of immune effectorcells under certain conditions (e.g., Kijimoto-Ochichai et al., CellMol. Life. Sci. 59:648 (2002); Davis et al., Curr. Top. Microbiol.Immunol. 266:85 (2002); Pawankar, Curr. Opin. Allerg. Clin. Immunol. 1:3(2001); Radaev et al., Mol. Immunol. 38:1073 (2002); Wurzburg et al.,Mol. Immunol. 38:1063 (2002); Sulica et al., Int. Rev. Immunol. 20:371(2001); Underhill et al., Ann. Rev. Immunol. 20:825 (2002); Coggeshall,Curr. Dir. Autoimm. 5:1 (2002); Mimura et al., Adv. Exp. Med. Biol.495:49 (2001); Baumann et al., Adv. Exp. Med. Biol. 495:219 (2001);Santoso et al., Ital. Heart J. 2:811 (2001); Novak et al., Curr. Opin.Immunol. 13:721 (2001); Fossati et al., Eur. J. Clin. Invest. 31:821(2001)), each of which is incorporated by reference herein in itsentirety.

Cells that are capable of mediating ADCC are examples of immune effectorcells. Other immune effector cells include Natural Killer cells,tumor-infiltrating T lymphocytes (TILs), cytotoxic T lymphocytes, andgranulocytic cells such as cells that comprise allergic responsemechanisms. Immune effector cells thus include, but are not limited to,cells of hematopoietic origin including cells at various stages ofdifferentiation within myeloid and lymphoid lineages and which may (butneed not) express one or more types of functional cell surface FcR, suchas T lymphocytes, B lymphocytes, NK cells, monocytes, macrophages,dendritic cells, neutrophils, basophils, eosinophils, mast cells,platelets, erythrocytes, and precursors, progenitors (e.g.,hematopoietic stem cells), as well as quiescent, activated, and matureforms of such cells. Other immune effector cells may include cells ofnon-hematopoietic origin that are capable of mediating immune functions,for example, endothelial cells, keratinocytes, fibroblasts, osteoclasts,epithelial cells, and other cells. Immune effector cells can alsoinclude cells that mediate cytotoxic or cytostatic events, or endocytic,phagocytic, or pinocytotic events, or that effect induction ofapoptosis, or that effect microbial immunity or neutralization ofmicrobial infection, or cells that mediate allergic, inflammatory,hypersensitivity and/or autoimmune reactions.

DBDpp as Albumin Fusions

Nucleic acid molecules encoding the DBDpp-albumin fusion proteins arealso encompassed herein, as are vectors containing these nucleic acids,host cells containing these nucleic acids vectors, and methods of makingthe DBDpp-albumin fusion proteins and using these nucleic acids,vectors, and/or host cells. The invention also encompassespharmaceutical formulations comprising a DBDpp-albumin fusion proteinand a pharmaceutically acceptable diluent or carrier. Such formulationscan be used in methods of treating, preventing, ameliorating ordiagnosing a disease or disease symptom in a patient, preferably amammal, most preferably a human, comprising the step of administeringthe pharmaceutical formulation to the patient.

DBDpp as Chimeric Receptors

In addition to the incorporation of DBD into soluble multi-domainproteins, the present invention provides a means by which to createcell-associated DBDpp, comprised of at least one DBDpp designed toimpart binding specificity a membrane bound fusion protein.DBDpp-receptors may be expressed by any cell type.

In one embodiment, the DBDpp-receptor fusion protein comprises achimeric antigen receptor (CAR), or DBDpp-CAR, composed of the followingelements: an extracellular targeting domain, a transmembrane domain anda cytoplasmic domain wherein the cytoplasmic domain comprises thesignaling domain. In another embodiment the DBDpp-CAR is composed of anextracellular targeting domain and a transmembrane domain. In a furtherembodiment the DBDpp-CAR is comprised of an extracellular domaincomposed of one or more DBDpp, in which each DBDpp constitutes atarget-specific binding domain with the same or different specificities.In several embodiments, the target-specific domain is directed to one(or more) of the cancer or tumor antigens disclosed herein, such asCD123, CD137, PD-L1, CD19, CD22, NY-ESO, or MAGE A3, as non-limitingexamples. In one embodiment, the intracellular domain (e.g., thecytoplasmic domain) of the DBDpp-CAR comprises the intracellular domainof CD3 zeta chain. In another embodiment the intracellular signalingdomain of the DBDpp is comprised of part of the intracellular domain ofCD3 zeta chain. In a further embodiment, the intracellular domain of theDBDpp-CAR comprises the intracellular domain of CD3 zeta chain and acostimulatory signaling region. The costimulatory signaling regionrefers to a portion of the DBDpp-CAR comprising all or part of theintracellular domain of a costimulatory molecule. Costimulatorymolecules are cell surface molecules other than antigens receptors ortheir ligands that are required for an efficient response of lymphocytesto antigen. Costimulatory molecules and portions of these molecules thatare able to confer costimulatory properties to a CAR are known in theart and can routinely be incorporated into the DBDpp-CAR. In addition,truncations or mutation to these intracellular signaling andcostimulatory domains may be incorporated to further enhance or reducereceptor signaling. In preferred embodiments, a T cell is geneticallymodified to stably express a DBDpp-CAR. In such embodiments thecytoplasmic domain of the DBDpp-CAR can be designed to comprise the CD28and/or 4-1BB signaling domain by itself or be combined with any otherdesired cytoplasmic domain(s) useful in the context of the invention. Inone embodiment, the cytoplasmic domain of the DBDpp-CAR can be designedto further comprise the signaling domain of CD3-zeta. For example, asdepicted schematically in FIG. 5B, in one embodiment, the DBDpp-CARcomprises an extracellular targeting domain, an extracellular proteinlinker with a transmembrane domain that passes through the cellularmembrane (such as found in T cells or NK cells), and a cytoplasmicdomain, optionally comprising multiple signaling modules. In severalembodiments, the DBDpp-CAR may also comprise an epitope tag. In severalembodiments, the cytoplasmic domain of the DBDpp-CAR can include but isnot limited to CD3-zeta, 4-1BB and CD28 signaling modules andcombinations thereof.

Extracellular Domain

Depending on the desired antigen to be targeted, the DBDpp-CAR can beengineered to include the appropriate antigen binding DBDpp that isspecific to the desired antigen target. For example, if CD19 is thedesired antigen that is to be targeted, one or more CD19-binding DBDppcan be incorporated into the target specific binding domain of theDBDpp-CAR. Alternatively DBDpp-CAR may include more than one DBDpp,imparting multi-specificity or multi-valency to the DBDpp-CAR.

The choice of DBDpp incorporated into the extracellular domain of theDBDpp receptor (e.g., DBDpp-CAR) depends upon the identity of the cellor cells to be targeted. For example, a DBDpp-CAR may specifically bindto cell surface proteins such as a receptor on the same cell or anothercell. In other embodiments, DBDpp-CAR specifically binds to a solublemolecule, such as an immunoglobulin. In other embodiments the targets ofinterest bound by the DBDpp-CAR include those associated with viral,bacterial and parasitic infections, diseases and disorders of the immunesystem (e.g., autoimmune disease).

In other embodiments a DBDpp-CAR may be chosen to recognize a ligandthat acts as a cell surface marker on target cells associated with acancer. A DBDpp-CAR can in some embodiments target and bind a tumorantigen (e.g., a TAA or other tumor antigen described herein orotherwise known in the art. Accordingly, provided herein are methods forcreating DBDpp-CAR, their use in creating chimeric cells such as, humanT cells and natural killer cells and the use of these chimeric T cellsin adoptive immunotherapy.

In the context provided herein, “tumor antigen” refers to antigens thatare common to specific hyperproliferative disorders such as cancer.Tumor antigens that can be specifically bound by a DBDpp in a DBDpp-CARare disclosed herein. In one embodiment, a DBDpp in a DBDpp-CARspecifically binds a tumor-specific antigen (TSA) or a tumor-associatedantigen (TAA). A TSA is unique to tumor cells and does not occur onother cells in the body. A TAA associated antigen is not unique to atumor cell and instead is also expressed on a normal cell underconditions that fail to induce a state of immunologic tolerance to theantigen. The expression of the antigen on the tumor may occur underconditions that enable the immune system to respond to the antigen. TAAsmay be antigens that are expressed on normal cells during fetaldevelopment when the immune system is immature and unable to respond orthey may be antigens that are normally present at extremely low levelson normal cells but which are expressed at much higher levels on tumorcells. Non-limiting examples of TSA or TAA antigens that can bespecifically bound by a DBDpp in a DBDpp-CAR includes a member selectedfrom: a differentiation antigen such as MART1/MelanA (MARTI), gp100(Pmel 17), tyrosinase, TRP1, TRP2; a tumor-specific multi-lineageantigen such as MAGE1, MAGE5, BAGE, GAGE1, GAGE2, pi5; an overexpressedembryonic antigen such as CEA; and overexpressed oncogene or mutatedtumor-suppressor gene such as p53, Ras, HER-2/neu; a unique tumorantigen resulting from chromosomal translocation such as BCR-ABL,E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; a viral antigen, such as the EpsteinBarr virus antigens EBVA and the human papillomavirus (HPV) antigens E6and E7; TSP-180, MAGE4, MAGE5, MAGE6, RAGE, NY-ESO, p185erbB2,p180erbB3, cmet, nm-23H1, PSA, TAG72, CA 19-9, CA72-4, CAM 17.1, NuMa,K-ras, beta-Catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4(791Tgp72)alpha-fetoprotem, beta-HCG, BCA225, BTAA, CA125, CA 15-3\CA 27.29\BCAA,CA195, CA242, CA50, CAM43, CD68\I, CO-029, FGF5, G250, Ga733VEpCAM,HTgp-175, M344, MA50, MG7-Ag, MOV 18, NB/70K, NY-CO-1, RCAS1, SDCCAG16,TA90\Mac-2, TAAL6, TAG72, TLP, and TPS; a glioma-associated antigen,carcinoembryonic antigen (CEA), β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulm, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinalcarboxylesterase, mut hsp70-2, MCSF, prostase, prostate-specific antigen(PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2/neu, survivinand telomerase, prostate-carcinoma tumor antigen-1 (PCTA1), MAGE, ELF2M,neutrophil elastase, ephrinB2, TACI (CD267), BAFF-R (CD268), BCMA(CD269), TLR4, insulin growth factor (IGF)I, IGFII, IGFI receptor andmesothelin.

In a particular embodiment, a DBDpp in the antigen binding moietyportion of a DBDpp-CAR specifically binds a target selected from: CD123,HVEM, BTLA, DR3, CD19, CD20, CD22, ROR 1, Mesothelin, CD33/1L3Ra, cMet,PSMA, Glycolipid F77, EGFRvIII, GD2, MY-ESO-1TCR, CD133, CD47 and MAGEA3 TCR. In another preferred embodiment, the DBDpp in the antigenbinding moiety portion of a DBDpp-CAR specifically bind all classes ofimmunoglobulin or specific isotypes, allotypes or idiotypes.

In one embodiment, a DBDpp in a DBDpp-CAR specifically binds a tumorantigen associated with a malignant tumor. Malignant tumors express anumber of tumor antigens that a DBDpp-CAR can be engineered to bind. Inone embodiment, a DBDpp of a DBDpp-CAR binds to an antigen selectedfrom: a tissue-specific antigen such as MART-1, tyrosinase and GP 100 inmelanoma and prostatic acid phosphatase (PAP) and prostate-specificantigen (PSA) in prostate cancer; a transformation-related molecule suchas the oncogene HER2/Neu ErbB2; an onco-fetal antigen such ascarcinoembryonic antigen (CEA); a B-cell lymphoma-specific idiotypeimmunoglobulin; a B-cell differentiation antigen such as CD19, CD20 andCD37; TSLPR and IL-7R on myeloid cells and cancer testis (CT) antigens(e.g. NY-ESO-1, LAGE-1a), CS-1, CD38, CD138, MUC1, HM1.24, CYP1B1, SP17,PRAME, Wilms' tumour 1 (WT1), and heat shock protein gp96 on multiplemyeloma cells.

Transmembrane Domain

“Transmembrane domain” (TMD) as used herein refers to the region of acell surface expressed DBDpp fusion protein such as a DBDpp-CAR, whichcrosses the plasma membrane. In some embodiments, the transmembranedomain of the DBDpp-CAR is the transmembrane region of a transmembraneprotein (for example Type I transmembrane proteins), an artificialhydrophobic sequence or a combination thereof. Other transmembranedomains will be apparent to those of skill in the art and may be used inconnection with alternate embodiments of the invention.

The DBDpp receptor (e.g., DBDpp-CAR) can be designed to contain atransmembrane domain that is fused to the extracellular domain of theDBDpp receptor. As described above, the fusion of the extracellular andtransmembrane domains can be accomplished with or without a linker. Inone embodiment, the transmembrane domain that is naturally associatedwith one of the domains in the DBDpp-CAR is used. In a specificembodiment, the transmembrane domain in the DBDpp-CAR is the CD8transmembrane domain. In some instances, the transmembrane domain of theDBDpp-CAR comprises the CD8 hinge domain. In some embodiments, thetransmembrane domain is be selected or modified by amino acidsubstitution to promote or inhibit association with other surfacemembrane proteins.

The transmembrane domain can be derived either from a natural or from asynthetic source. Where the source is natural, the domain can be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use for the purposes herein may be derived from (i.e.,comprise at least the transmembrane region(s) of) a member selected fromthe group: the alpha, beta or zeta chain of the T-cell receptor; CD28,CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64,CD80, CD86, CD134, CD137, and CD154. Alternatively the transmembranedomain can be synthetic, in which case the DBDpp-CAR transmembranedomain will comprise predominantly hydrophobic residues such as leucineand valine. In further embodiments, the transmembrane domain comprisesthe triplet of phenylalanine, tryptophan and valine at each end of asynthetic transmembrane domain.

“Extracellular spacer domain” (ESD) as used herein refers to thehydrophilic region which is between the antigen-specific targetingregion and the transmembrane domain. In some embodiments, the DBDpp-CARcomprise an extracellular spacer domain. In other embodiments, theDBDpp-CAR does not comprise an extracellular spacer domain. Theextracellular spacer domains include but are not limited to Fc fragmentsof antibodies or fragments or derivatives thereof, hinge regions ofantibodies or fragments or derivatives thereof, CH2 regions ofantibodies, CH3 regions of antibodies, artificial spacer sequences orcombinations thereof. Additional examples of extracellular spacerdomains include but are not limited to CD8a hinge, and artificialspacers made of polypeptides which may be as small as, for example, Gly3or CHI and CH3 domains of IgGs (such as human IgG4). In someembodiments, the extracellular spacer domain is any one or more of (i) ahinge, CH2 and CH3 regions of IgG4, (ii) a hinge region of IgG4, (iii) ahinge and CH2 of IgG4, (iv) a hinge region of CD8a, (v) a hinge, CH2 andCH3 regions of IgG1, (vi) a hinge region of IgG1 or (vi) a hinge and CH2region of IgG1. Other extracellular spacer domains will be apparent tothose of skill in the art and may be used in connection with alternateembodiments provided herein.

In some embodiments, a short oligo- or polypeptide linker, from about 1to 100 amino acids in length, is used to link together any of thedomains of a DBDpp-CAR. Linkers can be composed of flexible residueslike glycine and serine (or any other amino acid) so that the adjacentprotein domains are free to move relative to one another. The aminoacids sequence composition of the linker may be selected to minimizepotential immunogenicity of the DBDpp-CAR or DBDpp fusion protein.Longer linkers can be used when it is desirable to ensure that twoadjacent domains do not sterically interfere with one another. In someembodiments, preferably between 2 and 10 amino acids in length forms thelinkage between the transmembrane domain and the cytoplasmic signalingdomain of the DBDpp-CAR. In further embodiments, the linker is between10 and 15 amino acids in length, or between 15 and 20, or between 20 and30, or between 30 and 60, or between 60 and 100 amino acids in length(or any range in between those listed). In further embodiments, thelinker is a glycine-serine doublet sequence. Further embodiments employa fragment of the hinge region derived from the human T-cell surfaceglycoprotein CD8 alpha-chain (for example ranging from amino acidpositions 138 to 182 CD8 alpha chain; Swiss-Prot accession numberP01732). Further embodiments employ a fragment of the CD8 hinge regionthat has been further modified, through amino acid substitution, toimprove expression function or immunogenicity. Further embodimentsemploy a fragment of the extracellular region derived from the humanCD28 Further embodiments employ a fragment of the CD28 extracellularregion that has been further modified, through amino acid substitution,to improve expression function or immunogenicity.

Intracellular Domain

“Intracellular signaling domain” (ISD) or “cytoplasmic domain” as usedherein refer to the portion of the DBDpp-CAR which transduces theeffector function signal and directs the cell to perform its specializedfunction. The cytoplasmic domain (i.e., intracellular signaling domain)of a DBDpp-CAR is responsible for activation of at least one of thenormal effector functions of an immune cell engineered to express aDBDpp-CAR. The term “effector function” refers to a specialized functionof a cell. The effector function of a T cell, for example, includescytolytic activity and helper activity including the secretion ofcytokines. Thus the term “intracellular signaling domain” refers to theportion of a DBDpp-CAR protein which transduces the effector functionsignal and directs the cell to perform a specialized function. Whiletypically the entire intracellular signaling domain corresponding to anaturally occurring receptor can be employed, in many cases it is notnecessary to use the entire chain. To the extent that a truncatedportion of the intracellular signaling domain is used, such truncatedportion can be used in place of the intact chain as long as ittransduces the effector function signal. The term intracellularsignaling domain is thus meant to include any truncated portion of theintracellular signaling domain sufficient to transduce the effectorfunction signal. In one embodiment, an intracellular signaling domain inthe DBDpp-CAR includes the cytoplasmic sequences of the T cell receptor(TCR) and also the sequence of co-receptors that act in concert toinitiate signal transduction following antigen receptor engagement, orany derivative or variant of these sequences that has functionalcapability. Examples of domains that transduce an effector functionsignal include but are not limited to the ζ chain of the T-cell receptorcomplex or any of its homologs (e.g., η chain, FcsRly and β chains, MB 1(Iga) chain, B29 (Ig) chain, etc.), human CD3 zeta chain, CD3polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70,etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and othermolecules involved in T-cell transduction, such as CD2, CD5 and CD28.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of cytoplasmic signalingsequence: those that initiate antigen-dependent primary activationthrough the TCR (primary cytoplasmic signaling sequences) and those thatact in an antigen-independent manner to provide a secondary orco-stimulatory signal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs (ITAMs).

Examples of ITAM containing primary cytoplasmic signaling sequences thatare of particular use in the invention include those derived from TCRzeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD22,CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmicsignaling molecule in the CAR comprises a cytoplasmic signaling sequencederived from CD3 zeta.

“Co-stimulatory domain” (CSD) as used herein refers to the portion of aCAR or DBDpp-CAR which enhances the proliferation, survival and/ordevelopment of memory cells. The DBDpp-CAR may comprise one or moreco-stimulatory domains. Each co-stimulatory domain comprises thecostimulatory domain of any one or more of, for example, a member of theTNFR superfamily, selected from CD28, CD137 (4-1BB), CD134 (OX40),Dap10, CD27, CD2, CD5, ICAM-1, LFA-1(CD1 la/CD18), Lck, TNFR-I, TNFR-II,Fas, CD30, and CD40 or a combination thereof. Other co-stimulatorydomains (e.g., from other proteins) will be apparent to those of skillin the art and may be used in connection with alternate embodiments ofthe invention.

In a preferred embodiment, the cytoplasmic domain of a DBDpp-CARcomprises the CD3-zeta signaling domain by itself or combined with anyother desired cytoplasmic domain(s) useful in the context of theDBDpp-CAR. For example, the cytoplasmic domain of the DBDpp-CAR cancomprise a CD3 zeta chain portion and a costimulatory signaling region.The costimulatory signaling region refers to a portion of the CARcomprising the intracellular domain of a costimulatory molecule. Acostimulatory molecule is a cell surface molecule other than an antigenreceptor or their ligands that is required for an efficient response oflymphocytes to an antigen. Examples of such molecules include CD27,CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD1, ICOS, lymphocytefunction-associated antigen-1 (LFA1), CD2, CD7, LIGHT, NKG2C, B7H3,TIM1, and LAG-3.

Polypeptide linkers may be positioned between adjacent elements of theDBDpp-CAR. For example linkers may be positioned between adjacent DBDppor between DBDpp and the transmembrane domain or between thetransmembrane domain and the cytoplasmic domain or between adjacentcytoplasmic domains. The cytoplasmic signaling sequences within thecytoplasmic signaling portion of the DBDpp-CAR may be linked to eachother in a random or specified order. Optionally, a short linker,preferably between 2 and 10 amino acids in length may form the linkage.A glycine-serine doublet provides a particularly suitable linker.

Epitope Tag

In some embodiments, the DBDpp fusion protein comprises a peptideepitope tag. In some embodiments, the peptide tag is selected from thegroup consisting of a hexahistidyl (His6) tag, a myc tag and a FLAG tag.In additional embodiments, peptide tags include, but are not limited to,avitag (allows biotinylation of the tag and isolation withstreptavidin), calmodulin, E-tag, hemagglutinin (HA), S-tag, SBP-tag,softag 1, streptavidin, tetra or poly-cysteine, V5, VSV, and Xpress tag.Additionally polyhistidyl tags (other than 6 residues) can be used. Inadditional embodiments, covalent peptide tags, protein tags, and thelike can be used. Covalent peptide tags include, but are not limited to,isopeptag (covalently binds pilinC protein), Spytag (covalently binds tothe SpyCatcher protein), and Snooptag (covalently binds to theSnoopCatcher protein). In still additional embodiments, protein tags,including but not limited to biotin carboxyl carrier protein (BCCP),glutathione-s-transferase, green fluorescent protein (or otherfluorophore), Halo tag, Nus tag, thioredoxin, and Fc tags may optionallybe used. In still additional embodiments, multiple types of tags may beused. In still additional embodiments, no tag is used. Any combinationof extracellular, transmembrane and intracellular domains disclosedherein may be used, depending on the embodiment.

Linkers

The terms “linker” and spacer are used interchangeably herein to referto a peptide or other chemical linkage that functions to link otherwiseindependent functional domains. In one embodiment, a linker in a DBDppis located between a DBDpp and another polypeptide component containingan otherwise independent functional domain. Suitable linkers forcoupling the two or more linked DBDpp will be clear to the personsskilled in the art and may generally be any linker used in the art tolink peptides, proteins or other organic molecules. In particularembodiments, such a linker is suitable for constructing proteins orpolypeptides that are intended for pharmaceutical use.

Suitable linkers for operably linking a DBDpp and an additionalcomponent of a DBDpp fusion protein in a single-chain amino acidsequence include but are not limited to, polypeptide linkers such asglycine linkers, serine linkers, mixed glycine/serine linkers, glycine-and serine-rich linkers or linkers composed of largely polar polypeptidefragments.

In one embodiment, the linker is made up of a majority of amino acidsselected from glycine, alanine, proline, asparagine, glutamine, andlysine. In one embodiment, the linker is made up of a majority of aminoacids selected from glycine, alanine, proline, asparagine, asparticacid, threonine, glutamine, and lysine. In one embodiment, the DBDppfusion protein linker is made up of one or more of the amino acidsselected from glycine, alanine, proline, asparagine, glutamine, andlysine. In one embodiment, the DBDpp fusion protein linker is made up ofone or more of the amino acids selected from glycine, alanine, proline,asparagine, aspartic acid, threonine, glutamine, and lysine. In anotherembodiment, the DBDpp fusion protein linker is made up of a majority ofamino acids that are sterically unhindered. In another embodiment, alinker in which the majority of amino acids are glycine, serine, and/oralanine. In some embodiments, the peptide linker is selected frompolyglycines (such as (Gly)5 (SEQ ID NO: 188), and (Gly)8 (SEQ ID NO:189), poly(Gly-Ala), and polyalanines. In some embodiments, the peptidelinker contains the sequence of Gly-Gly-Gly-Gly-Thr-Gly-Gly-Gly-Gly-Ser(SEQ ID NO: 190). In some embodiments, the peptide linker contains thesequence of Gly-Gly-Gly-Gly-Asp-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 191).

In one embodiment, a DBDpp fusion comprises a DBDpp directly attached(i.e., without a linker) to another component of the DBDpp fusionprotein. In one embodiment, a DBDpp fusion comprises at least 2, atleast 3, at least 4, DBDpp directly attached to another component of theDBDpp fusion.

In another embodiment, a DBDpp can be operably linked to anothercomponent of a DBDpp fusion protein through a linker. DBDpp fusionproteins can contain a single linker, multiple linkers, or no linkers.In one embodiment, a DBDpp fusion comprises a DBDpp operably linked toanother component of the DBDpp fusion protein through a linker peptide.In one embodiment, a DBDpp fusion comprises at least 2, 3, 4, or 5 DBDoperably linked to another component of the DBDpp fusion protein througha linker peptide.

Linkers can be of any size or composition so long as they are able tooperably link a DBDpp in a manner that enables the DBDpp to bind atarget of interest. In some embodiments, linkers are about 1 to about100 amino acids, about 1 to 50 amino acids, about 1 to 20 amino acids,about 1 to 15 amino acids, about 1 to 10 amino acids, about 1 to 5 aminoacids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 to10 amino acids, or about 2 to 5 amino acids. It should be clear that thelength, the degree of flexibility and/or other properties of thelinker(s) may have some influence on the properties of the finalpolypeptide of the invention, including but not limited to the affinity,specificity or avidity for a target of interest, or for one or moreother target proteins of interest. When two or more linkers are used inthe DBDpp fusion proteins, these linkers may be the same or different.In the context and disclosure provided herein, a person skilled in theart will be able to routinely determine the optimal linker compositionand length for the purpose of operably linking a DBDpp and othercomponents of a DBDpp fusion protein.

The linker can also be a non-peptide linker such as an alkyl linker, ora PEG linker. For example, alkyl linkers such as —NH—(CH2)s-C(0)-,wherein s=2-20 can be used. These alkyl linkers may further besubstituted by any non-sterically hindering group such as lower alkyle.g., C1 C6) lower acyl, halogen (e.g., CI, Br), CN, NH2, phenyl, etc.An exemplary non-peptide linker is a PEG linker. In certain embodiments,the PEG linker has a molecular weight of about 100 to 5000 kDa, or about100 to 500 kDa.

Suitable linkers for coupling DBDpp and DBDpp fusion protein componentsby chemical cross-linking include, but are not limited to,homo-bifunctional chemical cross-linking compounds such asglutaraldehyde, imidoesters such as dimethyl adipimidate (DMA), dimethylsuberimidate (DMS) and dimethyl pimelimidate (DMP) orN-hydroxysuccinimide (NHS) esters such asdithiobis(succinimidylpropionate) (DSP) and dithiobis(sulfosuccinimidylpropionate) (DTSSP). Examples of suitable linkers forcoupling DBDpp and DBDpp fusion protein components ofhetero-bifunctional reagents for cross-linking include, but are notlimited to, cross-linkers with one amine-reactive end and asulfhydryl-reactive moiety at the other end, or with a NHS ester at oneend and an SH-reactive group (e.g., a maleimide or pyridyl).

In additional embodiments, one or more of the linkers in the DBDppfusion protein is cleavable. Examples of cleavable linkers include,without limitation, a peptide sequence recognized by proteases (in vitroor in vivo) of varying type, such as Tev, thrombin, factor Xa, plasmin(blood proteases), metalloproteases, cathepsins (e.g., GFLG, etc.), andproteases found in other corporeal compartments.

In one embodiment, the linker is a “cleavable linker” that facilitatesthe release of a DBDpp or cytotoxic agent in a cell. For example, anacid-labile linker (e.g., hydrazone), protease-sensitive (e.g.,peptidase-sensitive) linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari, Can. Res. 52:127-131 (1992); U.S.Pat. No. 5,208,020; U.S. Appl. Pub. No. 20090110753; each incorporatedby reference in their entireties) can be used wherein it is desirablethat the covalent attachment between a DBDpp or a cytotoxic agent andthe fusion partner is intracellularly cleaved when the composition isinternalized into the cell. The terms “intracellularly cleaved” and“intracellular cleavage” refer to a metabolic process or reaction insidea cell on an DBDpp drug conjugate whereby the covalent attachment, i.e.,linked via a linker between the DBDpp and cytotoxic agent, DBDpp andfusion partner, or between two DBDpp is broken, resulting in the freeDBDpp and/or cytotoxic agent dissociated inside the cell.

Linker optimization can be evaluated using techniques described hereinand/or otherwise known in the art. In some embodiments, linkers do notdisrupt the ability of a DBDpp to bind a target molecule and/or anotherDBDpp fusion protein component such as an antibody domain or fragment tobind an antigen.

DBDpp as Chemical Conjugates

DBDpp that promote specific binding to targets of interest can bechemically conjugated with a variety of compound such as fluorescentdyes, radioisotopes, chromatography compositions (e.g., beads, resins,gels, etc.) and chemotherapeutic agents. DBDpp conjugates have uses thatinclude but are not limited to purification, diagnostic, analytic,manufacturing and therapeutic applications.

The inherent lack of cysteines in the DBD sequence provides theopportunity for introduction of unique cysteines for purposes ofsite-specific conjugation.

In some embodiments, the DBDpp (e.g., a DBDpp fusion protein) containsat least one reactive residue. Reactive residues are useful, forexample, as sites for the attachment of conjugates such aschemotherapeutic drugs. The reactive residue can be, for example, acysteine, a lysine, or another reactive residue. Thus, a cysteine can beadded to a DBDpp at either the N or C terminus, or within the DBDppsequence. A cysteine can be substituted for another amino acid in thesequence of a DBDpp. In addition, a lysine can be added to a DBDpp ateither end or within the DBDpp sequence and/or a lysine can besubstituted for another amino acid in the sequence of a DBDpp. In oneembodiment, a reactive residue (e.g., cysteine, lysine, etc.) is locatedin a loop sequence of a DBD (e.g., Z₁ and Z₂ of SEQ ID NOS:7-11). In oneembodiment, a reactive residue is located between components of a DBDppfusion, e.g., in a linker located between a DBDpp and other component ofa DBDpp fusion protein. The reactive residue (e.g., cysteine, lysine,etc.) can also be located within the sequence of a DBDpp, or othercomponent of the DBDpp fusion protein. In one embodiment, a DBDpp or aDBDpp fusion protein comprises at least one, at least two, at leastthree reactive residues. In one embodiment, a DBDpp such as a DBDppfusion protein comprises at least one, at least two, or at least three,cysteine residues.

Production of DBDpp

The production of the DBDpp, useful in practicing the provided methods,may be carried out using a variety of standard techniques for chemicalsynthesis, semi-synthetic methods, and recombinant DNA methodologiesknown in the art. Also provided is a method for producing a DBDpp,individually or as part of multi-domain fusion protein, as solubleagents and cell associated proteins.

In several embodiments, the overall production scheme for DBDppcomprises obtaining a reference protein scaffold and identifying aplurality of residues within the scaffold for modification. Depending onthe embodiment, the reference scaffold may comprise a protein structurewith one or more alpha-helical regions, or other tertiary structure.Once identified, the plurality of residues can be modified, for exampleby substitution of an amino acid. In some embodiments substitution isconservative, while in other embodiments non-conservative substitutionsare made. In some embodiments a natural amino acid (e.g., one ofalanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, or valine) is substituted into the reference scaffold at thetargeted position for modification. In certain embodiments, themodifications do not include substituting in either a cysteine or aproline. After modifications have been made at all the identifiedpositions desired in a particular embodiment, the resulting modifiedpolypeptides (e.g., candidate DBDpp) can be recombinantly expressed, forexample in a plasmid, bacteria, phage, or other vector (e.g. to increasethe number of each of the modified polypeptides). The modifiedpolypeptides can then be purified and screened to identify thosemodified polypeptides that have specific binding to a particular targetof interest. In several embodiments, certain modified polypeptides willshow enhanced binding specificity for a target of interest vis-à-vis thereference scaffold, which in some embodiments may exhibit little or nobinding to a given target of interest. In additional embodiments,depending on the target of interest the reference scaffold may show someinteraction (e.g. nonspecific interaction) with a target of interest,while certain modified polypeptides will exhibit at least about twofold, at least about five fold, at least about 10 fold, at least about20 fold, at least about 50 fold, or at least about 100 fold (or more)increased binding specificity for the target of interest. Optionally,the reference sequence and/or the modified polypeptides (e.g., DBDpp)can be de-immunized. For example, residues or motifs that arepotentially immunogenic can be identified and modified in order toreduce or eliminate potential immune responses to the DBDpp. Additionaldetails regarding various embodiments of the production, selection, andisolation of DBDpp are provided in more detail below.

Recombinant Expression of DBDpp

In some embodiments, a DBDpp such as a DBDpp fusion protein is“recombinantly produced,” (i.e., produced using recombinant DNAtechnology). Exemplary recombinant methods available for synthesizingDBDpp fusion proteins, include, but are not limited to polymerase chainreaction (PCR) based synthesis, concatemerization, seamless cloning, andrecursive directional ligation (RDL) (see, e.g., Meyer et al.,Biomacromolecules 3:357-367 (2002), Kurihara et al., Biotechnol. Lett.27:665-670 (2005), Haider et al., Mol. Pharm. 2:139-150 (2005); andMcMillan et al., 32:3643-3646 (1999), the contents of each of which isherein incorporated by reference in its entirety).

Nucleic acids comprising a polynucleotide sequence encoding a DBDpp arealso provided. Such polynucleotides optionally further comprise, one ormore expression control elements. For example, the polynucleotide cancomprise one or more promoters or transcriptional enhancers, ribosomalbinding sites, transcription termination signals, and polyadenylationsignals, as expression control elements. The polynucleotide can beinserted within any suitable vector, which can be contained within anysuitable host cell for expression.

The expression of nucleic acids encoding DBDpp is typically achieved byoperably linking a nucleic acid encoding the DBDpp to a promoter in anexpression vector. Typical expression vectors contain transcription andtranslation terminators, initiation sequences, and promoters useful forregulation of the expression of the desired nucleic acid sequence.Methods known in the art can be used to routinely construct expressionvectors containing the nucleic acid sequence encoding a DBDpp along withappropriate transcriptional/translational control signals. These methodsinclude, but are not limited to in vitro recombinant DNA techniques,synthetic techniques and in vivo recombination/genetic recombination.The expression of the polynucleotide can be performed in any suitableexpression host known in the art including, but not limited to bacterialcells, yeast cells, insect cells, plant cells or mammalian cells. In oneembodiment, a nucleic acid sequence encoding a DBDpp is operably linkedto a suitable promoter sequence such that the nucleic acid sequence istranscribed and/or translated into DBDpp in a host. Promoters useful forexpression in E. coli, include but are not limited to, the T7 promoter.

In one embodiment, a vector comprising a DBDpp encoding nucleic acid isintroduced into a host cell (e.g., phagemid) for expression of a DBDpp.The vector can remain episomal or become chromosomally integrated, aslong as the insert encoding therapeutic agent can be transcribed.Vectors can be constructed by standard recombinant DNA technology.Vectors can be plasmids, phages, cosmids, phagemids, viruses, or anyother types known in the art, which are used for replication andexpression in prokaryotic or eukaryotic cells. It will be appreciated byone of skill in the art that a wide variety of components known in theart (such as expression control elements) can be included in suchvectors, including a wide variety of transcription signals, such aspromoters and other sequences that regulate the binding of RNApolymerase onto the promoter. Any promoter known or demonstrated to beeffective in the cells in which the vector will be expressed can be usedto initiate expression of DBDpp. Suitable promoters can be inducible(e.g., regulated) or constitutive. Non-limiting examples of suitablepromoters include the SV40 early promoter region, the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus, the HSV-1 (herpessimplex virus-1) thymidine kinase promoter, the regulatory sequences ofthe metallothionein gene, etc., as well as the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: elastase I gene control regionwhich is active in pancreatic acinar cells; insulin gene control regionwhich is active in pancreatic beta cells, mouse mammary tumor viruscontrol region which is active in testicular, breast, lymphoid and mastcells, albumin gene control region which is active in liver,alpha-fetoprotein gene control region which is active in liver, alpha1-antitrypsin gene control region which is active in the liver,beta-globin gene control region which is active in erythroid cells,myelin basic protein gene control region which is active inoligodendrocyte cells in the brain, myosin light chain-2 gene controlregion which is active in skeletal muscle, and gonadotropin releasinghormone gene control region which is active in the hypothalamus. In aparticular embodiment, the promoter is an immunoglobulin gene controlregion which is active in lymphoid cells.

In one embodiment, one or several nucleic acids encoding a DBDpp isexpressed under the control of a constitutive promoter or, alternately,a regulated expression system. Suitable regulated expression systemsinclude, but are not limited to, a tetracycline-regulated expressionsystem, an ecdysone inducible expression system, a lac-switch expressionsystem, a glucocorticoid-inducible expression system, atemperature-inducible promoter system, and a metallothioneinmetal-inducible expression system. If several different nucleic acidsencoding a DBDpp are contained within the host cell system, some of thenucleic acids may be expressed under the control of a constitutivepromoter, while others may be expressed under the control of a regulatedpromoter. Expression levels may be determined by methods known in theart, including Western blot analysis and Northern blot analysis.

A variety of host-expression vector systems can be utilized to express anucleic acid encoding a DBDpp. Vectors containing the nucleic acidsencoding the DBDpp (e.g., individual DBD subunits or DBDpp fusions) orportions or fragments thereof, include plasmid vectors, a single anddouble-stranded phage vectors, as well as single and double-stranded RNAor DNA viral vectors. Phage and viral vectors may also be introducedinto host cells in the form of packaged or encapsulated virus usingknown techniques for infection and transduction. Moreover, viral vectorsmay be replication competent or alternatively, replication defective.Alternatively, cell-free translation systems may also be used to producethe protein using RNAs derived from the DNA expression constructs (see,e.g., WO86/05807 and WO89/01036; and U.S. Pat. No. 5,122,464, eachincorporated in its entirety by reference herein).

Generally, any type of cells or cultured cell line can be used toexpress a DBDpp provided herein. In some embodiments the background cellline used to generate an engineered host cells is a phage, a bacterialcell, a yeast cell or a mammalian cell. A variety of host-expressionvector systems may be used to express the coding sequence a DBDpp fusionprotein. Mammalian cells can be used as host cell systems transfectedwith recombinant plasmid DNA or cosmid DNA expression vectors containingthe coding sequence of the target of interest and the coding sequence ofthe fusion polypeptide.

The cells can be primary isolates from organisms (including human),cultures, or cell lines of transformed or transgenic nature. In someembodiments the host cell is a human cell. In some embodiments, the hostcell is human T cell. In some embodiments, the host cell is derived froma human patient.

Useful host cells include but are not limited to microorganisms such as,bacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining DBDpp coding sequences; yeast (e.g., Saccharomyces, Pichia)transformed with recombinant yeast expression vectors containing DBDppcoding sequences; insect cell systems infected with recombinant virusexpression vectors (e.g., Baculovirus) containing DBDpp codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing DBDpp coding sequences. In particularembodiments, the mammalian cell systems are used to produce the DBDpp.Mammalian cell systems typically utilize recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

Prokaryotes useful as host cells in producing a DBDpp such as DBDppfusion protein, include gram negative or gram positive organisms suchas, E. coli and B. subtilis. Expression vectors for use in prokaryotichost cells generally contain one or more phenotypic selectable markergenes (e.g., genes encoding proteins that confer antibiotic resistanceor that supply an autotrophic requirement). Examples of usefulprokaryotic host expression vectors include the pKK223-3 (Pharmacia,Uppsala, Sweden), pGEM1 (Promega, Wis., USA), pET (Novagen, Wis., USA)and pRSET (Invitrogen, Calif., USA) series of vectors (see, e.g.,Studier, J. Mol. Biol. 219:37 (1991) and Schoepfer, Gene 124:83 (1993)).Exemplary promoter sequences frequently used in prokaryotic host cellexpression vectors include T7, (Rosenberg et al., Gene 56:125-135(1987)), beta-lactamase (penicillinase), lactose promoter system (Changet al., Nature 275:615 (1978)); and Goeddel et al., Nature 281:544(1979)), tryptophan (trp) promoter system (Goeddel et al., Nucl. AcidsRes. 8:4057, (1980)), and tac promoter (Sambrook et al., 1990, MolecularCloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.).

In one embodiment, a eukaryotic host cell systems is be used, includingyeast cells transformed with recombinant yeast expression vectorscontaining the coding sequence of a DBDpp, such as, the expressionsystems taught in U.S. Appl. No. 60/344,169 and WO03/056914 (methods forproducing humanlike glycoprotein in a non-human eukaryotic host cell)(the contents of each of which are incorporated by reference in theirentirety). Exemplary yeast that can be used to produce compositions ofthe invention, such as, DBD, include yeast from the genus Saccharomyces,Pichia, Actinomycetes and Kluyveromyces. Yeast vectors typically containan origin of replication sequence from a 2mu yeast plasmid, anautonomously replicating sequence (ARS), a promoter region, sequencesfor polyadenylation, sequences for transcription termination, and aselectable marker gene. Examples of promoter sequences in yeastexpression constructs include, promoters from metallothionein,3-phosphoglycerate kinase (Hitzeman, J. Biol. Chem. 255:2073 (1980)) andother glycolytic enzymes, such as, enolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phospho glycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Additional suitable vectors and promoters for use in yeastexpression as well as yeast transformation protocols are known in theart. See, e.g., Fleer, Gene 107:285-195 (1991) and Hinnen, PNAS 75:1929(1978).

Insect and plant host cell culture systems are also useful for producingthe compositions of the invention. Such host cell systems include forexample, insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus) containing the coding sequence of a DBD;plant cell systems infected with recombinant virus expression vectors(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the coding sequence of a DBD, including, but notlimited to, the expression systems taught in U.S. Pat. No. 6,815,184;U.S. Publ. Nos. 60/365,769, and 60/368,047; and WO2004/057002,WO2004/024927, and WO2003/078614, the contents of each of which isherein incorporated by reference in its entirety.

In an additional embodiment the host cell systems may be used, includinganimal cell systems infected with recombinant virus expression vectors(e.g., adenoviruses, retroviruses, adeno-associated viruses, herpesviruses, lentiviruses) including cell lines engineered to containmultiple copies of the DNA encoding a DBDpp either stably amplified(CHO/dhfr) or unstably amplified in double-minute chromosomes (e.g.,murine cell lines). In one embodiment, the vector comprising thepolynucleotide(s) encoding the DBDpp is polycistronic. Exemplarymammalian cells useful for producing these compositions include 293cells (e.g., 293T and 293F), CHO cells, BHK cells, NS0 cells, SP2/0cells, YO myeloma cells, P3X₆₃ mouse myeloma cells, PER cells, PER.C6(Crucell, Netherlands) cells VERY, Hela cells, COS cells, MDCK cells,3T3 cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT20cells, T47D cells, CRL7O30 cells, HsS78Bst cells, hybridoma cells, andother mammalian cells. Additional exemplary mammalian host cells thatare useful in practicing the invention include but are not limited, to Tcells. Some examples of expression systems and selection methods aredescribed in the following references and references cited therein:Borth et al., Biotechnol. Bioen. 71(4):266-73 (2000), in Werner et al.,Arzneimittelforschung/Drug Res. 48(8):870-80 (1998), Andersen et al.,Curr. Op. Biotechnol. 13:117-123 (2002), Chadd et al., Curr. Op,Biotechnol. 12:188-194 (2001), and Giddings, Curr. Op. Biotechnol.12:450-454 (2001). Additional examples of expression systems andselection methods are described in Logan et al., PNAS 81:355-359 (1984),Birtner et al. Methods Enzymol. 153:51-544 (1987)). Transcriptional andtranslational control sequences for mammalian host cell expressionvectors are frequently derived from viral genomes. Commonly usedpromoter sequences and enhancer sequences in mammalian expressionvectors include, sequences derived from Polyoma virus, Adenovirus 2,Simian Virus 40 (SV40), and human cytomegalovirus (CMV). Exemplarycommercially available expression vectors for use in mammalian hostcells include pCEP4 (Invitrogen) and pcDNA3 (Invitrogen).

Physical methods for introducing a nucleic acid into a host cell (e.g.,a mammalian host cell) include calcium phosphate precipitation,lipofection, particle bombardment, microinjection, electroporation, andthe like. Methods for producing cells comprising vectors and/orexogenous nucleic acids are well-known in the art. See, for example,Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York).

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian (e.g., human) cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362, the contents of each ofwhich is herein incorporated by reference in its entirety.

Methods for introducing a DNA and RNA polynucleotides of interest into ahost cell include electroporation of cells, in which an electrical fieldis applied to cells in order to increase the permeability of the cellmembrane, allowing chemicals, drugs, or polynucleotides to be introducedinto the cell. DBDpp containing DNA or RNA constructs may be introducedinto mammalian or prokaryotic cells using electroporation.

In a preferred embodiment, electroporation of cells results in theexpression of a DBDpp-CAR on the surface of T cells, NK cells, NKTcells. Such expression may be transient or stable over the life of thecell. Electroporation may be accomplished with methods known in the artincluding MaxCyte GT® and STX® Transfection Systems (MaxCyte,Gaithersburg, Md., USA).

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle). In the casewhere a non-viral delivery system is utilized, an exemplary deliveryvehicle is a liposome. The use of lipid formulations is contemplated forthe introduction of the nucleic acids into a host cell (in vitro, exvivo or in vivo). In another aspect, the nucleic acid can be associatedwith a lipid. The nucleic acid associated with a lipid can beencapsulated in the aqueous interior of a liposome, interspersed withinthe lipid bilayer of a liposome, attached to a liposome via a linkingmolecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they canbe present in a bilayer structure, as micelles, or with a “collapsed”structure. They can also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which can be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristoyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristoyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform may be used as the onlysolvent since it is more readily evaporated than methanol. “Liposome” isa generic term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,Glycobiology 5:505-510 (1991)). However, compositions that havedifferent structures in solution than the normal vesicular structure arealso encompassed. For example, the lipids can assume a micellarstructure or merely exist as non-uniform aggregates of lipid molecules.Also contemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell, or the presence of the recombinant nucleic acid sequence inthe host cell can routinely be confirmed through a variety of assaysknown in the art. Such assays include, for example, “molecularbiological” assays known in the art, such as Southern and Northernblotting, RT-PCR and PCR; “biochemical” assays, such as detecting thepresence or absence of a particular peptide, e.g., by immunologicalmeans (ELISAs and Western blots) or by assays described herein toidentify agents falling within the scope of the invention.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism, tissue, or cell and that encodes apolypeptide whose expression is manifested by some easily detectableproperty, e.g., enzymatic activity. Expression of the reporter gene isassayed at a suitable time after the DNA has been introduced into therecipient cells. A non-limiting list of suitable reporter genes caninclude genes encoding luciferase, beta-galactosidase, chloramphenicolacetyl transferase, secreted alkaline phosphatase, or the greenfluorescent protein gene (e.g., Ui-Tei et al., FEBS Lett. 479:79-82(2000)). Suitable expression systems are known in the art and can beprepared using known techniques or obtained commercially. In general,the construct with the minimal 5′ flanking region showing the highestlevel of expression of reporter gene is identified as the promoter. Suchpromoter regions can routinely be linked to a reporter gene and used toevaluate agents for the ability to modulate promoter-driventranscription.

A number of selection systems can be used in mammalian host-vectorexpression systems, including, but not limited to, the herpes simplexvirus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferaseand adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes, which can be employed in tk⁻, hgprt⁻ or aprt⁻ cells,respectively. Additionally, antimetabolite resistance can be used as thebasis of selection for e.g., dhfr, gpt, neo, hygro, trpB, hisD, ODC(ornithine decarboxylase), and the glutamine synthase system.

DBDpp Purification

Once a DBDpp such as a DBDpp fusion protein has been produced byrecombinant expression, it can be purified by any method known in theart for purification of a recombinant protein, for example, bychromatography (e.g., ion exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. In additionalembodiments, the DBDpp are optionally fused to heterologous polypeptidesequences described herein or otherwise known in the art to facilitatepurification. More particularly, it is envisioned that ligands (e.g.,antibodies and other affinity matrices) for DBDpp affinity columns foraffinity purification and that optionally, the DBDpp or other componentsof the DBDpp fusion composition that are bound by these ligands areremoved from the composition prior to final preparation of the DBDppusing techniques known in the art.

Expression of Cell Associated DBDpp

In another embodiment of the invention, production of DBDpp result incell associated DBDpp compositions. For example, the expression ofrecombinant vectors that encode DBDpp operably linked to a cell membraneanchor or transmembrane domain have the potential to remain cellassociated. DBDpp comprising chimeric antigen receptors areintentionally cell associated and used in the context of the cell inwhich they are expressed. One particular embodiment relates to astrategy of adoptive cell transfer of T cells which have been transducedto express a DBDpp chimeric antigen receptor (CAR). Preferably, the cellcan be genetically modified to stably express a DBDpp on its surface,conferring novel target specificity that is MHC independent.

A variety of viral-derived vectors can be used in applications in whichviruses are used for transfection and integration into a mammalian cellgenome. Viruses, which are useful as vectors include, but are notlimited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Lentiviral vectors are particularly suitableto achieving long-term gene transfer (e.g., adoptive T cell immunetherapy) since they allow long-term, stable integration of a transgeneand its propagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity. In general, a suitable vector contains an origin ofreplication functional in at least one organism, a promoter sequence,convenient restriction endonuclease sites, and one or more selectablemarkers, (e.g., WO 01/96584 and WO 01/29058; and U.S. Pat. No.6,326,193). Several vector promoter sequences are available forexpression of the transgenes. One example of a suitable promoter is theimmediate early cytomegalovirus (CMV) promoter sequence. This promotersequence is a strong constitutive promoter sequence capable of drivinghigh levels of expression of any polynucleotide sequence operativelylinked thereto. Another example of a suitable promoter is EF-1a.However, other constitutive promoter sequences can also be used,including, but not limited to the simian virus 40 (SV40) early promoter,mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV)long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemiavirus promoter, an Epstein-Barr virus immediate early promoter, a Roussarcoma virus promoter, as well as human gene promoters such as, but notlimited to, the actin promoter, the myosin promoter, the hemoglobinpromoter, and the creatine kinase promoter. Inducible promoters include,but are not limited to a metallothionein promoter, a glucocorticoidpromoter, a progesterone promoter, and a tetracycline promoter.

In order to assess the expression of a DBDpp-CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors, in other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Prior to expansion and genetic modification of the T cells of theinvention, a source of T cells is obtained from a subject. T cells canbe obtained from a number of sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, and tumors. In certain embodiments provided herein, anynumber of T cell lines available in the art, may be used.

A full discussion of T cell isolation, culturing, activation andexpansion methods may be found in WO 2012079000, the contents of whichis herein incorporated by reference in its entirety.

Additionally provided is a host cell comprising nucleic acids encoding aDBDpp described herein. Further provided is a composition comprising anucleic acid sequence encoding the DBDpp.

“Co-express” as used herein refers to simultaneous expression of two ormore protein coding sequences. The coding sequences may be nucleic acidsencoding, for example, a single protein or a chimeric protein as asingle polypeptide chain.

Chemical Synthesis of DBDpp

In addition to recombinant methods, DBDpp production may also be carriedout using organic chemical synthesis of the desired polypeptide using avariety of liquid and solid phase chemical processes known in the art.Various automatic synthesizers are commercially available and can beused in accordance with known protocols. See, for example, Tam et al.,J. Am. Chem. Soc, 105:6442 (1983); Merrifield, Science 232:341-347(1986); Barany and Merrifield, The Peptides, Gross and Meienhofer, eds,Academic Press, New York, 1-284; Barany et al., Int. J. Pep. ProteinRes., 30:705 739 (1987); Kelley et al. in Genetic Engineering Principlesand Methods, Setlow, J. K., ed. Plenum Press, N Y. 1990, vol. 12, pp.1-19; Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co.,San Francisco, 1989. One advantage of these methodologies is that theyallow for the incorporation of non-natural amino acid residues into thesequence of the DBDpp.

The DBDpp that are used in the methods of the present invention may bemodified during or after synthesis or translation, e.g., byglycosylation, acetylation, benzylation, phosphorylation, amidation,pegylation, formylation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, ubiquitination, etc. (See, e.g.,Creighton, Proteins: Structures and Molecular Properties, 2d Ed. (W.H.Freeman and Co., N.Y., 1992); Postranslational Covalent Modification ofProteins, Johnson, ed. (Academic Press, New York, 1983), pp. 1-12;Seifter, Meth. Enzymol., 182:626-646 (1990); Rattan, Ann. NY Acad. Sci.,663:48-62 (1992).) In specific embodiments, the peptides are acetylatedat the N-terminus and/or amidated at the C-terminus.

Any of numerous chemical modifications may be carried out by knowntechniques, including, but not limited to acetylation, formylation, etc.Additionally, the derivative may contain one or more non-classical aminoacids.

Populations of DBDpp can be Represented by Libraries of Polypeptides

A “library” of DBDpp refers to a plurality of unique DBDpp. A “vectorlibrary” of DBDpp refers to a plurality of unique nucleic acids encodingDBDpp. These libraries of DBDpp can be used to select for and identifysequences that promote binding to specific predetermined targets.

In one embodiment, DBDpp are represented by a mixed population, orlibrary, of different DBDpp molecules. A library of DBDpp does not implyany particular size limitation to the number unique polypeptidemolecules. A library can contain as few as 3, 5, 6, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 75, or 100 unique DBDpp, and can range togreater than 1020 different DBDpp. In some embodiments the library hasup to about 10⁴, 10⁵, 10⁶, 10⁷, or 10⁸ unique DBDpp. In furtherembodiments the library has up to about 10¹² different DBDpp.

In one embodiment, a population of polypeptide variants is based on asequence of core residues and variant residues. For example, SEQ ID NO:3variant residues are denoted by an X, where X can be any amino acidresidue independent of the identity of any other residue denoted X inthe sequence. In certain embodiments, X can comprise a null position(e.g., no amino acid at that site). In the scaffold amino acid sequencethe different varied amino acids X may be chosen from all 20 naturallyoccurring amino acid residues in such a way that any of these 20naturally occurring amino acid residues may be present at thecorresponding X position in any given variant. The selection of aminoacid residue in each position is more or less randomized, depending onthe embodiment. It is also possible to limit the group from which thedifferent varied amino acid residues are selected to 19, 18, 17, 16 orless of the 20 naturally occurring amino acid residues. For example, insome embodiments, the variant residues are not replaced by cysteineand/or proline. The variability in different positions can be adjustedindividually, between one, meaning no randomization, up to all 20 aminoacids. Random introduction of a smaller subset of amino acids may beobtained by careful selection of the deoxyribonucleotide basesintroduced, for example the codons T(A/C)C may be introduced to obtain arandom introduction of either serine or tyrosine at a given position inthe polypeptide chain. Likewise, the codons (T/C/A/G)CC can beintroduced to obtain a random introduction of phenylalanine, leucine,alanine and valine at a given position in the polypeptide chain. Aswould be understood by a person of ordinary skill in the art manyalternatives of deoxyribonucleotide base combinations can be used toobtain different combinations of amino acids at a given position in thepolypeptide chain. The set of amino acids that can appear at a givenposition in the polypeptide chain can also be determined by theintroduction of trinucleotides during the oligonucleotide synthesis,instead of one deoxyribonucleotide base at a time.

Also provided is a library containing a plurality of DBDpp. In someembodiments, the DBDpp library comprises a plurality of different DBDppthat comprise the amino acid sequence of SEQ ID NO:1 wherein 5 to 25, 5to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 aminoacid residues have been modified; and wherein the DBDpp specificallybinds a target of interest. In some embodiments, 5 to 25, 5 to 30, 5 to35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 of the modified aminoacid residues are substitutions. In some embodiments, 5 to 25, 5 to 30,5 to 35, 5 to 40, 5 to 45, or 5 to 50 of the modified amino acidresidues are conservative substitutions. In some embodiments, 5 to 25, 5to 30, 5 to 35, 5 to 40, 5 to 45, or 5 to 50 of the modified amino acidresidues are non-conservative substitutions. In a further embodiment, 5to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of theamino acid residue modifications are conservative substitutions and 5 to15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of the aminoacid residue modifications are non-conservative substitutions. Inadditional embodiments, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5to 50, 5 to 55, or 5 to 60 of the substitutions are at amino acidresidues of SEQ ID NO:1 selected from the group consisting of: M1, G2,S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21,G22, G23, S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39,S40, E41, Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55,R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72, andN73. In a further embodiment, 1 to 20, 1 to 30, or 1 to 40 of thesubstitutions are at amino acid residues of SEQ ID NO:1 selected fromthe group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15,T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41,Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68,A69, and Y70. In another embodiment, the library comprises at least 2,3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp thatspecifically binding different targets. In a further embodiment, thedifferent targets bound by DBDpp in the library are selected from thegroup consisting of: a nucleic acid, an oligosaccharide, a peptide, aprotein, a cell surface antigen, and a small organic molecule. In afurther embodiment, the library comprises at least 2, 3, 4, 5, 10, 25,50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind aprotein target selected from the group consisting of: an immunoglobulin,an enzyme, a hormone, a serum protein, a cell surface protein, atherapeutic protein, a TSA, a CSA, and a protein containing a sequencetag. In a further embodiment, the library comprises at least 2, 3, 4, 5,10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specificallybind a target disclosed herein. In an additional embodiment, the libraryis a vector library or a host cell library. In an additional embodiment,the vector library is a library of host cells. In another embodiment,the host cell library comprises a plurality of host cells that displaythe DBDpp on their surface. In a further embodiment, the host cells arephage that display the DBDpp on their surface.

In some embodiments, the DBDpp library comprises: (a) 3 DBDpp thatspecifically bind to different targets; (b) 3, 4, 5, 6, 7, 8, 9, 10, ormore than 10 DBDpp having different sequences that specifically bind tothe same target; (c) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpphaving different sequences that specifically bind to the same epitope ofa target of interest; (d) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpphaving different sequences that specifically bind to different epitopesof a target; or (e) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpphaving different sequences that compete for binding to the same target.

Also provided is a library containing a plurality of DBDpp. In someembodiments, the DBDpp library comprises a plurality of different DBDppthat comprise the amino acid sequence of SEQ ID NO:1 wherein 5 to 25, 5to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 aminoacid residues have been modified; and wherein the DBDpp specificallybinds a target of interest. In some embodiments, 5 to 25, 5 to 30, 5 to35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 of the modified aminoacid residues are substitutions. In some embodiments, 5 to 25, 5 to 30,5 to 35, 5 to 40, 5 to 45, or 5 to 50 of the modified amino acidresidues are conservative substitutions. In some embodiments, 5 to 25, 5to 30, 5 to 35, 5 to 40, 5 to 45, or 5 to 50 of the modified amino acidresidues are non-conservative substitutions. In a further embodiment, 5to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of theamino acid residue modifications are conservative substitutions and 5 to15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of the aminoacid residue modifications are non-conservative substitutions. Inadditional embodiments, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5to 50, 5 to 55, or 5 to 60 of the substitutions are at amino acidresidues of SEQ ID NO:1 selected from the group consisting of: M1, G2,S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21,G22, G23, S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39,S40, E41, Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55,R57, K58, E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72, andN73. In a further embodiment, 1 to 20, 1 to 30, or 1 to 40 of thesubstitutions are at amino acid residues of SEQ ID NO:1 selected fromthe group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15,T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41,Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68,A69, and Y70. In another embodiment, the library comprises at least 2,3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp thatspecifically binding different targets. In a further embodiment, thedifferent targets bound by DBDpp in the library are selected from thegroup consisting of: a nucleic acid, an oligosaccharide, a peptide, aprotein, a cell surface antigen, and a small organic molecule. In afurther embodiment, the library comprises at least 2, 3, 4, 5, 10, 25,50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind aprotein target selected from the group consisting of: an immunoglobulin,an enzyme, a hormone, a serum protein, a cell surface protein, atherapeutic protein, a TSA, a CSA, and a protein containing a peptidetag. In a further embodiment, the library comprises at least 2, 3, 4, 5,10, 25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specificallybind a target disclosed herein. In an additional embodiment, the libraryis a vector library or a host cell [including viral particles] library.In an additional embodiment, the vector library is a library of hostcells. In another embodiment, the host cell library comprises aplurality of host cells that display the DBDpp on their surface. In afurther embodiment, the host cells are phage that display the DBDpp ontheir surface.

In some embodiments, the DBDpp library comprises: (a) 3, 4, 5, 6, 7, 8,9, 10, or more than 10 DBDpp that specifically bind to differenttargets; (b) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp havingdifferent sequences that specifically bind to the same target; (c) 3, 4,5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences thatspecifically bind to the same epitope of a target of interest; (d) 3, 4,5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences thatspecifically bind to different epitopes of a target; or (e) 3, 4, 5, 6,7, 8, 9, 10, or more than 10 DBDpp having different sequences thatcompete for binding to the same target.

In an additional embodiment, the DBDpp library contains a plurality ofdifferent nucleic acid sequences encoding DBDpp, that comprise the aminoacid sequence of SEQ ID NO:1 wherein a total of 5 to 25, 5 to 30, 5 to35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 amino acid residueshave been modified; and wherein the DBDpp specifically binds a target ofinterest. In another embodiment, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5to 45, 5 to 50, 5 to 55, or 5 to 60 of the modified amino acid residuesencoded by the nucleic acids sequences are substitutions. In anotherembodiment, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, or 5 to 50 ofthe modified amino acid residues are conservative substitutions. Inanother embodiment, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, or 5 to50 of the encoded modified amino acid residues are non-conservativesubstitutions. In a further embodiment, 5 to 15, 5 to 20, 5 to 25, 5 to30, 5 to 35, 5 to 40, or 5 to 45 of the encoded amino acid residuemodifications are conservative substitutions and 5 to 15, 5 to 20, 5 to25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of the encoded amino acidresidue modifications are non-conservative substitutions. In additionalembodiments, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to55, or 5 to 60 of the encoded substitutions are at amino acid residuesof SEQ ID NO:1 selected from the group consisting of: Ml, G2, S3, W4,A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23,S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41,Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58,E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72, and N73. In afurther embodiment, 1 to 20, 1 to 30, or 1 to 40 of the encodedsubstitutions are at amino acid residues of SEQ ID NO:1 selected fromthe group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15,T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41,Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68,A69, and Y70. In a further embodiment, the nucleic acids optionallyencode a DBDpp that further comprises an amino acid sequence wherein 1to 5, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45of the residues corresponding to the solvent inaccessible residues ofthe amino acid sequence of SEQ ID NO:1 are substituted and wherein theDBDpp specifically binds a target of interest. In another embodiment,the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10,25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specificallybind different targets. In a further embodiment, the different targetsbound by DBDpp in the library are selected from the group consisting of:a nucleic acid, an oligosaccharide, a peptide, a protein, a cell surfaceantigen, and a small organic molecule. In a further embodiment, thelibrary comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25,50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind aprotein target selected from the group consisting of: an immunoglobulin,an enzyme, a hormone, a serum protein, a cell surface protein, atherapeutic protein, a TSA, a CSA, and a protein containing a peptidetag. In a further embodiment, the library comprises nucleic acidsencoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000different DBDpp that specifically bind a target disclosed herein. In anadditional embodiment, the vector library is contained in host cells(e.g., viral particles). In another embodiment, the library comprises aplurality of host cells that display the DBDpp on their surface. In afurther embodiment, the host cells are phage that display the DBDpp ontheir surface. In some embodiments, the DBDpp library comprises: (a) 3,4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp that specifically bind todifferent targets; (b) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpphaving different sequences that specifically bind to the same target;(c) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having differentsequences that specifically bind to the same epitope of a target; (d) 3,4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequencesthat specifically bind to different epitopes of a target; or (e) 3, 4,5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences thatcompete for binding to the same target.

Nucleic acids encoding DBDpp such as DBDpp fusion proteins are alsoprovided. In some embodiments the host cell containing the nucleic acidsis a bacteria, yeast, fungal or mammalian cell. In a further embodiment,the host cell is an immune cell. In a further embodiment, the host cellis a human immune cell. In a further embodiment, the human immune cellexpresses the DBDpp on its cell surface. In particular embodiments, thenucleic acid encode a DBDpp fusion protein. In a further embodiment, thehost cell expresses the DBDpp as a fusion protein on the cell surface.Additionally provided herein are vector libraries comprising a pluralityof nucleic acids encoding the DBDpp.

In one embodiment, a vector library comprises a plurality of differentnucleic acids encoding DBDpp, wherein the encoded DBDpp comprises anamino acid sequence selected from the group consisting of: (a)MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆L QAYRHN(SEQ ID NO:4), wherein X₅, X₈, X₉, X₁₂, X₁₅, X₁₆, X₁₉, X₅₅, X₅₈, X₅₉,X₆₂, X₆₅, and X₆₆, is a natural and/or non-natural amino acid residue;(b) MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2), wherein X₅, X₆, X₉, X₁₀, X₁₃, X₁₆, X₁₇,X₃₀, X₃₃, X₃₄, X₃₇, X₄₀, X₄₁, and X₄₄, is a natural and/or non-naturalamino acid residue; (c)MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN (SEQ ID NO:3), wherein X₃₂, X₃₃,X₃₆, X₃₉, X₄₀, X₄₃, X₅₇, X₅₈, X₆₁, X₆₄, X₆₅, and X₆₈, is a naturaland/or non-natural amino acid residue, and; (d)MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN (SEQ ID NO:5),wherein X₅, X₆, X₉, X₁₀, X₁₃, X₁₆, X₁₇, X₃₂, X₃₃, X₃₆, X₃₉, X₄₀, X₄₃,X₅₅, X₅₈, X₅₉, X₆₂, X₆₅, and X₆₆, is a natural and/or non-natural aminoacid residue; and (e) MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN(SEQ ID NO:6), wherein X₅, X₈, X₉, X₁₂, X₁₅, X₁₆, X₁₉, X₃₀, X₃₃, X₃₄,X₃₇, X₄₀, X₄₁, X₄₄, X₅₇, X₅₈, X₆₁, X₆₄, X₆₅, and X₆₈, is a naturaland/or non-natural amino acid residue; and wherein the DBDppspecifically binds a target of interest. In an additional embodiment,X_(n) is a natural amino acid residue. In a further embodiment, X_(n) isa natural amino acid residue other than cysteine or proline. In anadditional embodiment, a plurality of the vectors in the library encodea DBDpp fusion protein. In another embodiment, the library comprisesnucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250,500, or 1000 different DBDpp that specifically bind different targets.In a further embodiment, the different targets bound by DBDpp encoded bythe nucleic acids in the library are selected from the group consistingof: a nucleic acid, an oligosaccharide, a peptide, a protein, a cellsurface antigen, and a small organic molecule. In a further embodiment,the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10,25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specificallybind a protein target selected from the group consisting of: animmunoglobulin, an enzyme, a hormone, a serum protein, a cell surfaceprotein, a therapeutic protein, a TSA, a CSA, and a protein containing apeptide tag. In a further embodiment, the library comprises nucleicacids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or1000 different DBDpp that specifically bind a target disclosed herein.In an additional embodiment, a plurality of the vectors of the vectorlibrary are contained in host cells (e.g., viral particles such asphage), E. coli, yeast, and mammalian cells. In another embodiment, thehost cells display DBDpp on their surface. In a further embodiment, thehost cells are phage that display DBDpp on their surface. In someembodiments, the vector library comprises: (a) nucleic acids encoding 3,4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp that specifically bind todifferent targets; (b) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10,or more than 10 DBDpp 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpphaving different sequences that specifically bind to the same target;(c) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10DBDpp having different sequences that specifically bind to the sameepitope of a target; (d) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10,or more than 10 DBDpp having different sequences that specifically bindto different epitopes of a target; (e) nucleic acids encoding 3, 4, 5,6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences thatcompete for binding to the same target; or (f) 3, 4, 5, 6, 7, 8, 9, 10,or more than 10 different nucleic acids encoding the same DBDpp. Hostcells containing the vectors are also provided.

In one embodiment, a vector library comprises a plurality of nucleicacids encoding DBDpp comprising an amino acid sequence selected from thegroup consisting of: (a)MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAAFEKEIAAFESELQAYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN (SEQ ID NO:9), wherein X₅, X₈, X₉, X₁₂, X₁₅,X₁₆, X₁₉, X₅₀, X₅₃, X₅₄, X₅₇, X₆₀, and X₆₁, is a natural and/ornon-natural amino acid residue, and Z₁ and Z₂ is 2 to natural and/ornon-natural amino acid residues; (b) MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7), wherein X₅, X₆, X₉, X₁₀, X₁₃, X₁₆, X₁₇, X₂₈,X₃₁, X₃₂, X₃₅, X₃₈, X₃₉, and X₄₂, is a natural and/or non-natural aminoacid residue, and Z₁ and Z₂ is 2 to 30 natural and/or non-natural aminoacid residues; (c) MGSWAEFKQRLAAIKTRLEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN (SEQID NO:8), wherein X₃₀, X₃₁, X₃₄, X₃₇, X₃₈, X₄₁, X₅₂, X₅₃, X₅₆, X₅₉, X₆₀,and X₆₃, is a natural and/or non-natural amino acid residue, and Z₁ andZ₂ is 2 to 30 natural and/or non-natural amino acid residues; (d)MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇₁RX₆₀X₆₁LQAYRHN(SEQ ID NO:10), wherein X₅, X₆, X₉, X₁₀, X₁₃, X₁₆, X₁₇, X₃₀, X₃₁, X₃₄,X₃₇, X₃₈, X₄₁, X₅₀, X₅₃, X₅₄, X₅₇, X₆₀, and X₆₁, is a natural and/ornon-natural amino acid residue, and Z₁ and Z₂ is 2 to 30 natural and/ornon-natural amino acid residues; and (e) MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN(SEQ ID NO:11), wherein X₅, X₈, X₉, X₁₂, X₁₅, X₁₆, X₁₉, X₂₈, X₃₁, X₃₂,X₃₅, X₃₈, X₃₉, X₄₂, X₅₂, X₅₃, X₅₆, X₅₉, X₆₀, and X₆₃, is a naturaland/or non-natural amino acid residue, and Z₁ and Z₂ is 2 to 30 naturaland/or non-natural amino acid residues; and wherein the DBDppspecifically binds a target of interest. In an additional embodiment,X_(n) is a natural amino acid residue. In a further embodiment, X_(n) isa natural amino acid residue other than cysteine or proline. In anadditional embodiment, a plurality of the vectors in the library encodea DBDpp fusion protein. In another embodiment, the library comprisesnucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250,500, or 1000 different DBDpp that specifically bind different targets.In a further embodiment, the different targets bound by DBDpp encoded bythe nucleic acids in the library are selected from the group consistingof: a nucleic acid, an oligosaccharide, a peptide, a protein, a cellsurface antigen, and a small organic molecule. In a further embodiment,the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10,25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specificallybind a protein target selected from the group consisting of: animmunoglobulin, an enzyme, a hormone, a serum protein, a cell surfaceprotein, a therapeutic protein, a TSA, a CSA, and a protein containing apeptide tag. In a further embodiment, the library comprises nucleicacids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or1000 different DBDpp that specifically bind a target disclosed herein.In an additional embodiment, a plurality of the vectors of the vectorlibrary are contained in host cells. In another embodiment, the hostcells (e.g., viral particles) display DBDpp on their surface. In afurther embodiment, the host cells are phage that display DBDpp on theirsurface. In some embodiments, the vector library comprises: (a) nucleicacids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp thatspecifically bind to different targets; (b) nucleic acids encoding 3, 4,5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences thatspecifically bind to the same target; (c) nucleic acids encoding 3, 4,5, 6, 7, 8, 9, 10, or more than 10 DBDpp having different sequences thatspecifically bind to the same epitope of a target; (d) nucleic acidsencoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having differentsequences that specifically bind to different epitopes of a target; (e)nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpphaving different sequences that compete for binding to the same target;or (f) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different nucleic acidsequences encoding the same DBDpp sequence. Host cells containing thevectors are also provided.

In some embodiments, 4, 5, 10 or more DBDpp encoded by the nucleic acidsin the library specifically bind different targets.

In one embodiment, a vector library comprises comprising a plurality ofdifferent nucleic acid sequences encoding DBDpp, that comprise the aminoacid sequence of SEQ ID NO:1 wherein a total of 5 to 25, 5 to 30, 5 to35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, or 5 to 60 amino acid residueshave been modified; and wherein the DBDpp specifically binds a target ofinterest. In another embodiment, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5to 45, 5 to 50, 5 to 55, or 5 to 60 of the modified amino acid residuesencoded by the nucleic acids sequences are substitutions. In anotherembodiment, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, or 5 to 50 ofthe modified amino acid residues are conservative substitutions. Inanother embodiment, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, or 5 to50 of the encoded modified amino acid residues are non-conservativesubstitutions. In a further embodiment, 5 to 15, 5 to 20, 5 to 25, 5 to30, 5 to 35, 5 to 40, or 5 to 45 of the encoded amino acid residuemodifications are conservative substitutions and 5 to 15, 5 to 20, 5 to25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45 of the encoded amino acidresidue modifications are non-conservative substitutions. In additionalembodiments, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to55, or 5 to 60 of the encoded substitutions are at amino acid residuesof SEQ ID NO:1 selected from the group consisting of: Ml, G2, S3, W4,A5, E6, K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, L21, G22, G23,S24, E25, A26, E27, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41,Q43, A44, Y45, K46, G47, K48, G49, N50, P51, E52, E54, A55, R57, K58,E59, A61, A62, R64, D65, E66, Q68, A69, Y70, R71, H72, and N73. In afurther embodiment, 1 to 20, 1 to 30, or 1 to 40 of the encodedsubstitutions are at amino acid residues of SEQ ID NO:1 selected fromthe group consisting of: G2, S3, W4, A5, E6, K8, Q9, R10, A12, A13, K15,T16, R17, E19, A20, A29, A30, E32, K33, E34, A36, A37, E39, S40, E41,Q43, A44, E52, E54, A55, R57, K58, E59, A61, A62, R64, D65, E66, Q68,A69, and Y70. In a further embodiment, the nucleic acids optionallyencode a DBDpp that further comprises an amino acid sequence wherein 1to 5, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, or 5 to 45of the residues corresponding to the solvent inaccessible residues ofthe amino acid sequence of SEQ ID NO:1 are substituted and wherein theDBDpp specifically binds a target of interest. In another embodiment,the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10,25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specificallybind different targets. In a further embodiment, the different targetsbound by DBDpp in the library are selected from the group consisting of:a nucleic acid, an oligosaccharide, a peptide, a protein, a cell surfaceantigen, and a small organic molecule. In a further embodiment, thelibrary comprises nucleic acids encoding at least 2, 3, 4, 5, 10, 25,50, 75, 100, 250, 500, or 1000 different DBDpp that specifically bind aprotein target selected from the group consisting of: an immunoglobulin,an enzyme, a hormone, a serum protein, a cell surface protein, atherapeutic protein, a TSA, a CSA, and a protein containing a peptidetag. In a further embodiment, the library comprises nucleic acidsencoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or 1000different DBDpp that specifically bind a target disclosed herein. In anadditional embodiment, the vector library is contained in host cells(e.g., viral particles). In another embodiment, the library comprises aplurality of host cells that display the DBDpp on their surface. In afurther embodiment, the host cells are phage that display the DBDpp ontheir surface. In some embodiments, the vector library comprises: (a)nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDppthat specifically bind to different targets; (b) nucleic acids encoding3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having differentsequences that specifically bind to the same target; (c) nucleic acidsencoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having differentsequences that specifically bind to the same epitope of a target; (d)nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpphaving different sequences that specifically bind to different epitopesof a target; or (e) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, ormore than 10 DBDpp having different sequences that compete for bindingto the same target; or (f) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10different nucleic acids encoding the same DBDpp. Host cells containingthe vectors are also provided.

In some embodiments, the vector library comprises: (a) nucleic acidsencoding 3 DBDpp that specifically bind to different targets ofinterest; (b) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or morethan 10 DBDpp having different sequences that specifically bind to thesame target of interest; (c) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9,10, or more than 10 DBDpp having different sequences that specificallybind to the same epitope of a target of interest; (d) nucleic acidsencoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having differentsequences that specifically bind to different epitopes of a target ofinterest; (e) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or morethan 10 DBDpp having different sequences that compete for binding to thesame target of interest; or (f) 3 different nucleic acid sequencesencoding the same DBDpp sequence.

In one embodiment, the vector library comprises a plurality of nucleicacids encoding DBDpp comprising an amino acid selected from the groupconsisting of: (a)MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAAFEKEIAAFESELQAYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN (SEQ ID NO:9), wherein X₅, X₈, X₉, X₁₂, X₁₅, X₁₆,X₁₉, X₅₀, X₅₃, X₅₄, X₅₇, X₆₀, and X₆₁, is a natural and/or non-naturalamino acid residue, and Z₁ and Z₂ is 2 to 30 natural and/or non-naturalamino acid residues; (b) MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7), wherein X₅, X₆, X₉, X₁₀, X₁₃, X₁₆, X₁₇, X₂₈, X₃₁,X₃₂, X₃₅, X₃₈, X₃₉, and X₄₂, is a natural and/or non-natural amino acidresidue, and Z₁ and Z₂ is 2 to 30 natural and/or non-natural amino acidresidues; (c) MGSWAEFKQRLAAIKTRLEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX41AYZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN(SEQ ID NO:8), wherein X₃₀, X₃₁, X₃₄, X₃₇, X₃₈, X₄₁, X₅₂, X₅₃, X₅₆, X₅₉,X₆₀, and X₆₃ is a natural and/or non-natural amino acid residue, and Z₁and Z₂ is 2 to 30 natural and/or non-natural amino acid residues; (d)MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN (SEQ IDNO:10), wherein X₅, X₆, X₉, X₁₀, X₁₃, X₁₆, X₁₇, X₃₀, X₃₁, X₃₄, X₃₇, X₃₈,X₄₁, X₅₀, X₅₃, X₅₄, X₅₇, X₆₀, and X₆₁, is a natural and/or non-naturalamino acid residue, and Z₁ and Z₂ is 2 to 30 natural and/or non-naturalamino acid residues; and (e) MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN (SEQ ID NO:11), wherein X₅, X₈, X₉, X₁₂, X₁₅, X₁₆, X₁₉, X₂₈, X₃₁,X₃₂, X₃₅, X₃₈, X₃₉, X₄₂, X₅₂, X₅₃, X₅₆, X₅₉, X₆₀, and X₆₃, is a naturaland/or non-natural amino acid residue, and Z₁ and Z₂ is 2 to 30 naturaland/or non-natural amino acid residues; and wherein the DBDppspecifically binds a target of interest. In an additional embodiment,X_(n) is a natural amino acid residue. In a further embodiment, X_(n) isa natural amino acid residue other than cysteine or proline. In anadditional embodiment, a plurality of the vectors in the library encodea DBDpp fusion protein. In another embodiment, the library comprisesnucleic acids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250,500, or 1000 different DBDpp that specifically bind different targets.In a further embodiment, the different targets bound by DBDpp encoded bythe nucleic acids in the library are selected from the group consistingof: a nucleic acid, an oligosaccharide, a peptide, a protein, a cellsurface antigen, and a small organic molecule. In a further embodiment,the library comprises nucleic acids encoding at least 2, 3, 4, 5, 10,25, 50, 75, 100, 250, 500, or 1000 different DBDpp that specificallybind a protein target selected from the group consisting of: animmunoglobulin, an enzyme, a hormone, a serum protein, a cell surfaceprotein, a therapeutic protein, a TSA, a CSA, and a protein containing apeptide tag. In a further embodiment, the library comprises nucleicacids encoding at least 2, 3, 4, 5, 10, 25, 50, 75, 100, 250, 500, or1000 different DBDpp that specifically bind a target disclosed herein.In an additional embodiment, a plurality of the vectors of the vectorlibrary is contained in host cells (including viral particles). Inanother embodiment, the host cells (e.g., viral particles) display DBDppon their surface. In a further embodiment, the host cells are phage thatdisplay DBDpp on their surface. In some embodiments, at least two,three, four, five, or ten of the DBDpp encoded in the vector libraryspecifically bind different targets. In some embodiments, the DBDppbinds a target of interest selected from the group consisting of: anucleic acid, an oligosaccharide, a peptide, a protein, a cell surfaceantigen, and a small organic molecule. In a further embodiment, theDBDpp target of interest is a protein selected from the group consistingof: an immunoglobulin, an enzyme, a hormone, a serum protein, a cellsurface protein, a therapeutic protein, a TSA, a CSA, and a proteincontaining a peptide tag. In a further embodiment, the DBDppspecifically binds a target disclosed herein. Also provided is a libraryof host cells (e.g., viral particles) containing the vector library. Insome embodiments, the library contains a plurality of host cells (e.g.,viral particles) that display the DBDpp on their surface. In particularembodiments, the host cells are phage that display the DBDpp on theirsurface. In some embodiments, the vector library comprises: (a) nucleicacids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp thatspecifically bind to different targets of interest; (b) nucleic acidsencoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having differentsequences that specifically bind to the same target of interest; (c)nucleic acids encoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpphaving different sequences that specifically bind to the same epitope ofa target of interest; (d) nucleic acids encoding 3, 4, 5, 6, 7, 8, 9,10, or more than 10 DBDpp having different sequences that specificallybind to different epitopes of a target of interest; (e) nucleic acidsencoding 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DBDpp having differentsequences that compete for binding to the same target of interest; or(f) 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different nucleic acidsequences encoding the same DBDpp sequence.

It is envisioned that the DBDpp can be modified to tailor thepolypeptides to the specific use intended, without departing from thescope provided herein. Such modifications may comprise additional aminoacids at the N- or C-terminus of the DBDpp and/or labels or therapeuticagents that are chemically conjugated or otherwise bound to the DBDpp.The additional amino acid residues discussed above may also constituteone or more polypeptide domain(s) with any desired function, such asanother binding function, or an enzymatic function, or a metal ionchelating function, or a fluorescent function, or mixtures thereof.

Selecting, Isolating and Identifying DBDpp

Methods for selecting, isolating and identifying DBDpp that specificallybind a target of interest from a plurality of DBDpp, such as those in alibrary, are also provided. In one embodiment, a method of screening alibrary of DBDpp for binding with a binding partner, comprises: (a)obtaining a population displaying a library of DBDpp; (b) contacting thepopulation with the target of interest under conditions suitable forbinding; and (c) identifying those DBDpp that bind to the target. Twoexemplary DBDpp display selection processes include panning andcell-based screening selection.

In illustrative examples provided herein, DBDpp phage display librariesare prepared and screened for DBDpp having desired properties, includingthe ability to specifically bind numerous validated therapeutic anddiagnostic targets. Representative DBDpp identified in these screens arefurther characterized and demonstrated to display desirable propertiesuseful in for example, purification, diagnostic, and therapeuticapplications.

Display Library

As described herein, substitutions in the reference scaffold of SEQ IDNO:1 provide a versatile molecular recognition platform. Such DBDpp canbe used in methods for preparing libraries of DBDpp which can bescreened against targets of interest. Such screening methods can be usedto identify DBDpp with desired properties such as the ability to bind atarget of interest. The population of DBDpp used in the selection oftarget-specific DBDpp can be in different forms, and can be but are notlimited to protein libraries, nucleic acid libraries, vector librariesand host cell libraries.

Various methods known in the art for preparing modifications of nucleicacid can be used to prepare (encode) DBDpp having modification in one ormore amino acid residues compared to another DBDpp and/or the referencescaffold of SEQ ID NO:1. Nucleic acids encoding DBDpp may be obtainedusing standard methods in the art, such as chemical synthesis,recombinant methods and/or obtained from biological sources. Nucleicacid of interest may be placed under the control of one or more elementsnecessary for their expression in any particular host cell. A variety ofhost cells are available to propagate nucleic acids encoding DBDpp, anddisplay methods are known in the art and described herein that may beused in display DBDpp on their surface. Display methods include withoutlimitation phage display, bacterial display, yeast display, ribosomedisplay, and mRNA display.

In some embodiments, the generation of a (partially) randomized DBDpplibrary requires the (partial) randomization of specific positionswithin the reference scaffold sequence of SEQ ID NO:1. In additionalembodiments, other reference sequences may be used and modifiedaccording to the methods disclosed herein. In one embodiment, a DBDpplibrary for use in the methods provided herein are generated byrecombinant DNA techniques. In particular, libraries of nucleic acidsequences encoding DBDpp each differing in sequence at particular aminoacid positions can be obtained by site-directed or random mutagenesis ofa template sequence. Random amino acid residues can be introduced atspecific positions in an amino acid sequence using techniques known inthe art such as selecting (introducing) ‘NNK’ or ‘NNS’ codons atcorresponding positions in the nucleotide sequence encoding said aminoacid sequence. Methods for producing such libraries are known in the artand commercial services are available for generating such libraries. Thenucleotide(s) determining the relevant amino acid residues in thepositions of interest are mutated in different ways such as to obtain alibrary of sequences encoding different DBDpp.

Libraries are optionally created through the selective or randommutation of specific solvent exposed amino acid sequence positions ofthe DBD.

In some embodiments, the number of substituted amino acid residuepositions in DBDpp libraries provided herein range from 5 to 20 aminoacid residue positions. Thus, a defined set of substituted amino acidresidue positions in a DBDpp library provided herein comprise 5 to 20defined substituted amino acid residue positions, such as 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, defined substituted aminoacid residue positions. In several embodiments, the substituted aminoacid residues are natural or non-natural amino acids. In severalembodiments, any of the 20 natural amino acids can be used. However, insome embodiments, the substitutions do not result in the replacement ofany amino acids with a cysteine and/or a proline.

A library of DBDpp can contain any suitable number of different DBDppsequences. In some embodiments, the library of DBDpp contain least 2, atleast 5, at least 10, at least 50, at least 100, at least 1000, at least10,000, at least 105, at least 106, at least 107, at least 108, at least109 or more different DBDpp sequences (e.g., DBDpp fusion proteins).

The notion “substituted amino acid residue position”, when referring toa library of different-DBDpp sequences, refers to an amino acid residueposition at which at least two different amino acid residue types arelocated when at least two of the amino acid sequences of the differentDBDpp from a library of DBDpp are compared to each other.

In one embodiment, the disclosure encompasses methods of producing alibrary (i.e., a collection or plurality) of DBDpp which differ fromeach other in at least one of a defined set of 5 to 20 substituted aminoacid residue positions. Therefore, the sequences within a library ofDBDpp differ from each other at any one or more particular amino acidpositions that are comprised in a selected, defined, or random set.Accordingly, the term “different sequences” or “different DBDppsequences” refers to the occurrence of sequence variation or sequencedifferences in a defined set of amino acid residue positions between twoor more DBDpp in a library.

Display Vehicle

The population or library of molecules is displayed on a typical displayvehicle (e.g., bacteriophage, E. coli, ribosome) that affords thecoupling of phenotype to genotype.

In some embodiments, the DBDpp of the library are displayed on thesurface of a phage particle, a ribosome, a bacterium, a yeast cell, amammalian cell or any other suitable (micro)organism, so as tofacilitate screening or selection to isolate the desired DBDpp sequenceshaving detectable binding affinity for, or detectable in vitro activityon the target of interest. A major advantage of this technology is thecoupling of genotype (i.e., the encapsulated DNA encoding the displayedprotein) and phenotype (i.e., the displayed protein such as a DBDppprovided herein) which allows affinity-based selection from librarieswith millions to trillions of polypeptide variants in a relativelysimple in vitro assay.

Suitable methods, techniques and host organisms for displaying andselecting or screening a library of substituted DBDpp sequences ornucleotide sequences encoding such substituted DBDpp sequences, andwhich are applicable to DBDpp having desired features, are known to theperson skilled in the art. Such methods are described, for example, inGeorgiou, Nat. Biotechnol. 15:29-34 (1997); Wittrup, Curr. Opin.Biotechnol. 12:395-399 (2001); Lipovsek and Pluckthun, J Immunol Methods290:51-67 (2004); Reiersen, Nucl Acids Res, 33:e10, 2005; Levin, MolBioSyst, 2:49-57 (2006); Bratkovic, Cell. Mol. Life. Sci. 67:749-767(2010). For example, the technology of phage library display, and theselection by means of a phage display technique may be chosen as amethod for high-throughput identification of protein-specific binders,because it is one of the most robust and versatile selection techniquesavailable (Scott, Science 249:386-390 (1990); Bratkovic, Cell. Mol. LifeSci. 67:749-767 (2010)).

Additionally, display technology can be used to alter, e.g., improve thebinding properties of DBDpp. See, for example, Scott, Science 249: 386(1990); Devlin, Science 249: 404 (1990); U.S. Pat. Nos. 5,223,409,5,733,731, 5,498,530, 5,432,018, 5,338,665, and 5,922,545; WO 96/40987and WO 98/15833, the contents of each of which is herein incorporated byreference in its entirety. In peptide phage display libraries, naturaland/or non-naturally occurring peptide sequences can be displayed byfusion with coat proteins of filamentous phage. The displayed peptidescan be affinity-eluted against a target of interest if desired. Theretained phage can be enriched by successive rounds of affinitypurification and repropagation. The best binding DBDpp can be sequencedto identify key residues and mutagenesis libraries may be created andscreened to further optimize the sequence of the best binders. Lowman,Ann. Rev. Biophys. Biomol. Struct. 26: 401-24 (1997).

Phage Display

A typical phage display protocol involves the use of a filamentous phage(phagemid) surface expression system, production of phage particles in abacterial host with each particle displaying the gene product of onemember of the gene library as a fusion with one type of its coatproteins (gIII or gVIII proteins). A library of phage particles is takenthrough a selection process for binding to an immobilized targetmolecule (‘biopanning’) involving binding of the phage library to thetarget, washing steps to remove non-bound phage DBDpp, and elution ofbound particles. Usually several rounds of panning are necessary toselect molecules with the desired characteristics involvingreamplification of eluted phage in the bacterial host and selection onthe immobilized target.

For example, using a phagemid display (Kay et al., Phage Display ofPeptides and Proteins. A Laboratory Manual, B. K. Kay et al. 1996) agiven DBDpp library may be represented by a collection of phagemids eachof which encodes for a fusion protein comprising a member of the DBDpplibrary fused to the minor coat protein pIII. These phagemids can beintroduced into suitable E. coli cells (e.g. TG1) by electroporation orother means. Using infection with helper phage, phage are produced(packaging also the phagemid genome) that display the DBDpp-fusionprotein. These phage can be used to select binders against a giventarget and the selected phage can be propagated by infecting E. coli TG1(Stratagene).

Thus, in particular embodiments, the DBDpp libraries are provided as aphage library and binding DBDpp are identified by contacting the phagewith the labeled target of interest, after which binding phages areretrieved by detection or selective collection of the labeled, boundtarget. In one embodiment, a biotinylated target is used, whereby phagewhich generate a DBDpp that specifically binds to the target arecaptured with a streptavidin-coated support (e.g., magnetic beads). Insome embodiments, the selection steps of the methods for producing oneor more DBDpp having detectable binding affinity for a target ofinterest, may comprise the (further) enrichment of the DBDpp library orthe mixture of DBDpp libraries for DBDpp having detectable bindingaffinity for the target of interest by iterative execution of the stepsof contacting a target of interest with a DBDpp library or with amixture of DBDpp libraries (including a plurality of DBDpp) of theinvention and subsequently identifying from the DBDpp library or mixtureof DBDpp libraries being contacted with the protein, one or more DBDpphaving detectable binding affinity for the target of interest. The stepof selecting a DBDpp that has detectable in vitro activity byinteracting with a target of interest may comprises: (a) contacting aDBDpp library or a mixture of DBDpp libraries of the invention with thecytokine or growth factor or cytokine or growth factor receptor ofinterest, and (b) identifying from the DBDpp library or mixture of DBDpplibraries, the one or more DBDpp having detectable in vitro activity onthe target of interest.

In illustrative embodiments disclosed herein in the Examples, phagedisplay methods are used to display and screen DBDpp for the ability tospecifically bind a target of interest

It is demonstrated herein that the DBD domain can be displayed andselected on the surface of phage. Different libraries of DBDpp, based onthe scaffold of SEQ ID NO:1, and described herein in the examples, wereprepared and subjected to phage display methods to demonstrate thatDBDpp can be produced that specifically bind to different targets ofinterest including CD137, CD47, CTLA4, DR5, KIR, PD-L1, PD1 and TIM3.

Cell Display

In some embodiments, the library screening techniques include a cellsurface display system. The cell surface display system may compriseprokaryotic cells, such as Gram+ cells, or eukaryotic cells, such asyeast cells. Numerous cell surface display systems are known in the artand can routinely be adapted for screening DBDpp libraries. Prokaryoticsystems are, for example, described in Francisco et al., PNAS90:10444-10448 (1993) and Lee et al., Trends Biotechnol 21:45-52 (2003).Eukaryotic systems are described for example in Boder et al., Nat.Biotechnol. 15:553-557 (1997) and Gal et al., Curr. Opin. Struct. Biol.17:467-473 (2007). “E. coli display” methods such aspeptidoglycan-associated lipoprotein (PAL) fusion are also encompassedherein. For example, a DBDpp peptide can be fused to the carboxylterminus of the lac repressor and expressed in E. coli.

The bacterial display and yeast display technologies known in the artallow expression of recombinant proteins on the surface of yeast cellsS. cerevisiae (Boder, Nat. Biotechnol. 15:553-557 (1997) or bacteria (E.coli, Staphylococcus carnosus) (Daugherty., 1998, Wernerus, Appl.Environ. Microbiol. 69(9):5328-5335 (2003)) as a fusion with thea-agglutinin yeast adhesion receptor or a bacterial outer membraneprotein (OMP) respectively.

In some embodiments, the expressed fusion proteins also contain apeptide tag allowing quantification of the library surface expression byflow cytometry. Combined with indirect fluorescent labeling of theligand, anti-tag labeling allows cell sorting by FACS (fluorescenceactivated cell sorting) and the determination of the binding affinitiesof the interactions (Feldhaus et al., Nat. Biotechnol. 21:163-70 (2003);Wernerus et al. Appl. Environ. Microbiol. 69(9):5328-35 (2003)).

In Vitro Display

In vitro (also known as cell-free or acellular) display methods may alsobe employed to select for, isolate and identify DBDpp that bind a targetof interest. In one example, translation of random RNA is halted priorto ribosome release, resulting in a library of polypeptides with theirassociated RNA still attached. This and related methods are collectivelyreferred to as “ribosome display.” Other known methods employ chemicallinkage of peptides to RNA. See, for example, Roberts et al., PNAS94:12297-303 (1997). This and related methods are collectively referredto as “RNA-peptide screening, RNA display and mRNA display.”Alternatively, in vitro display methods may employ DNA as the geneticcomponent to which the expressed polypeptide is coupled. A method knownas cis-display affords the in vitro selection of peptides from librariesof protein—DNA complexes and is described in U.S. Pat. No. 7,842,476 B2,the contents of which are herein incorporated by reference in itsentirety. Chemically derived peptide libraries have been developed inwhich peptides are immobilized on stable, non-biological materials, suchas polyethylene rods or solvent-permeable resins. Another chemicallyderived peptide library uses photolithography to scan peptidesimmobilized on glass slides. These and related methods are collectivelyreferred to as “chemical-peptide screening.” Chemical-peptide screeningmay be advantageous in that it allows use of D-amino acids and otherunnatural analogues, as well as non-peptide elements. Both biologicaland chemical methods are reviewed in Wells, Curr. Opin. Biotechnol.3:355-362 (1992).

Selection of DBDpp

Biopanning is a known iterative selection and screening method to enrichan initial population of different molecules (such as a DBDpp library)for molecules having an affinity for a target of choice. Library membersthat have affinity for the target are allowed to bind. Non-specificallyor weakly bound members are washed from the support. Then the boundlibrary members are recovered (e.g., by elution) from the support.Recovered library members are collected for further analysis (e.g.,screening) or pooled for an additional round of selection.

In one embodiment, the target is captured on the solid support afterincubation with the phage library. The immobilization of the target canbe performed by many different methods known in the art. Examples ofsolid support are microtiter plates or tubes (e.g. Maxisorp plates,Maxisorp tubes, Nunc) or magnetic beads (Dynabead®, Invitrogen). Thetarget can either be directly coated on the plastic or the beads(surface activated Dynabeads, e.g. Dynabeads M270 Epoxy, Invitrogen) orvia streptavidin when the target is biotinylated (e.g. Dynabeads MyOneStreptavidin T1, Invitrogen).

In addition the target may be bound non-covalently to the bead via anintermediate affinity molecule such as an antibody or protein A directedagainst the target or a target-associated peptide tag. Peptide tags suchas His-tags or alternatively, an antibody directed against the targetcan also be used to capture the target on the support. These alternativepeptide tags are also compatible with the Dynabeads (Dynabeads His-tagisolation and pull down, Invitrogen) and Protein A or Protein G coupledDynabeads (Dynabeads-Protein A/G, Invitrogen). To immobilize the targeton magnetic beads, the recommendations of the manufacturer are followedfor each specific bead type.

The capturing step may then consist of trapping the target to coatedmagnetic beads, thereby capturing indirectly phage bound to the target.The target-phage interaction is performed in solution. To be able towash away the non-binding phage, the target needs to be immobilized on asolid support. The immobilization of the target in the solublebiopanning method is identical to the immobilization possibilities inthe direct biopanning protocols.

A classical biopanning protocol consists of 2, 3 to 5 or more selectionrounds, depending on the type of target and library. Each selectionround consists of typically different steps: (1) immobilization of thetarget of choice to a support. This step is optional, as biopanning canalso be performed in a format wherein the target is not-immobilized butkept in solution (in case of soluble target) or remains anchored on acell (in case of e.g. a membrane anchored target such as a receptor),(2) incubation of the library with the target, (3) washing steps toeliminate non-specific binders, (4) optionally elution of the bindersand (5) amplification of the eluted binders from step (4) or from step(3) (in case step (4)) was omitted in consecutive screening rounds). Thesteps 1 to 5 will be repeated two, three, four or more times to isolatefrom the initial library target-specific binders. After the biopanning,the target—specificity of the binders isolated from the differentselection rounds is typically analyzed in ELISA assays or similarassays.

In one embodiment, a method of screening for DBDpp that specificallybind a target of interest comprises the steps of: (a) contacting atarget of interest with a plurality of DBDpp; and (b) identifying aDBDpp that specifically binds the target of interest. The step ofcontacting the target of interest with the plurality of DBDpp may beaffected in any way known in the art. For example, in one embodiment,the target of interest is immobilized on a solid support and contactinga solution containing the plurality of DBDpp molecules with theimmobilized target of interest. Such a procedure is akin to an affinitychromatographic process, with the affinity matrix being comprised of theimmobilized target of interest. The DBDpp having a selective affinityfor the target of interest can then be purified using techniques knownin the art, such as affinity selection. The composition of the solidsupport, process for attaching the target of interest to the solidsupport, and the reagents, conditions and methods of screening for andisolating DBDpp having a selective affinity for a target of interest arelargely conventional and known to those of ordinary skill in the art. Incertain situations, it may be desirable to wash away any unbound DBDppfrom a mixture of the target of interest and/or one or more DBDpp boundto the target of interest prior to attempting to determine or to detectthe presence of a selective affinity interaction. Such a wash step maybe particularly desirable when the target of interest is bound to asolid support.

It will be understood that the selection step of the methods describedherein can be performed by way of a method commonly known as a selectionmethod or a by way of a method commonly known as a screening method.Both methods envisage the identification and subsequent isolation (e.g.,the selection step) of desirable components (e.g., DBDpp librarymembers) from an original ensemble comprising both desirable andnon-desirable components (e.g., a DBDpp library). In the case of aselection method, library members will typically be isolated by a stepwherein the desired property is applied to obtain the desired goal; insuch case, the desired property is usually restricted to the property ofa high affinity for a given target interest. Such method is generallyknown as an affinity selection method and, such affinity selectionmethod will be applied to a DBDpp library for the purpose of selectingDBDpp having a high affinity for a target of interest. In additionalembodiments, the library is screened DBDpp having desired kineticproperties such as high on-rate for binding to a given target ofinterest, or low off-rate for library members bound to said target byadjusting the appropriate selection conditions (e.g. short incubationtimes or long wash cycles, or other conditions as is known by someoneskilled in the art of library selection techniques). Alternatively, inthe case of a screening method, library members will typically beisolated by a step wherein all library members, or at least asubstantial collection of library members, are individually examinedwith respect to a given desired property, and wherein members havingsuch desired property are retained whereas members not having suchdesired property are discarded; in such case, and in the contextprovided herein, desired properties may relate to either a high affinityfor a target of interest, or a functional activity such as, theinhibition, reduction and/or prevention of the activity of a target ofinterest. Accordingly, it is submitted that the selection step of themethods may be accomplished by either an (affinity) selection techniqueor by an affinity-based or activity-based functional screeningtechnique, both techniques resulting in the selection of one or moreDBDpp having beneficial (favorable, desirable, superior) affinity oractivity properties compared to the non-selected DBDpp of the DBDpp.

Screening of DBDpp

After selection, the identified members of the library can beindividually isolated and screened.

A screening differs from a selection in that a screen is characterizedby the analysis of library members individually (or in pools) whereas aselection is characterized by analysis of library members that areseparated from other members during the process (e.g., retained, eluted,or washed off). In one embodiment, a collection of library members isdirectly screened, without being subjected to a selection step. Thisapproach, for example, can be used during affinity maturation protocolsthat are known and can be routinely applied.

The ability of a DBDpp to specifically bind a target of interest can bedetermined using or routinely modifying assays and other methodologiesdescribed herein or otherwise known in the art. For example,DBDpp-target interaction can be assayed as described in the Examplesbelow or alternatively, using in vitro or in vivo binding assays such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, fluorescentimmunoassays, protein A immunoassays, immunohistochemistry (IHC) andBIAcore analysis. Similarly, the ability of a DBDpp to specifically binda target of interest and to alter the biological activity of the targetcan be determined using or routinely modifying assays and othermethodologies described herein or otherwise known in the art. Assaysevaluating the ability of a DBDpp to functionally affect its target(e.g., assays to measure signaling, proliferation, migration etc.) canalso be used to indirectly assess DBDpp-target interaction.Additionally, DBDpp can be identified based on their effects in assaysthat measure particular pathways or activities. For example, assays thatmeasure signaling pathways (e.g., phosphorylation studies ormultimerization), ion channel fluxes, intracellular cAMP levels,cellular activities such as migration, adherence, proliferation, orapoptosis, and viral entry, replication, budding, or integration can beused to identify, characterize, and improve the desired properties ofthe DBDpp. The ability of a DBDpp to competitively inhibit anotherDBDpp-containing sequence can be determined using techniques known inthe art, including ELISA and BIAcore analysis.

Identification of DBDpp

Where a DBDpp candidate contained in a library of DBDpp, is displayed ona suitable cell or phage or particle, the nucleic acid coding sequencecan be isolated and routinely determined. It is possible to isolate fromsaid cell or phage or particle, the nucleotide sequence that encodesthat DBDpp sequence. In this way, the nucleotide sequence of theselected DBDpp library member(s) can be determined by routine sequencingmethods.

DBDpp library members that are specific for the target can becharacterized by nucleic acid sequencing. Sequence information is usedto classify the members and to remove redundant members (i.e., membersthat encode that same DBDpp). DBDpp libraries and library members(including some members in which epitope tags have been added) accordingto several embodiments include, but are not limited to those identifiedin below in Table 1. Additionally included are those DBDpp thatcorrespond to any of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11,wherein one or more of the X_(n) positions are substituted with anatural or non-natural amino acid. In some embodiments, the DBDpp thatcorrespond to any of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 donot include cysteine and/or proline residues that are substituted intoan X_(n) position.

TABLE 1 Non-limiting Examples of DBDpp Libraries and Library Members SEQID NO Sequence Target Library   2MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇ F1FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALRKEAAAIRDELQAYRHN   3MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀EL F2X₄₃AYKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN   4MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAAFEKEIAAFESEL F3QAYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN   5MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAAFX₃₂X₃₃EIX₃₆AF C1X₃₉X₄₀ELX₄₃AYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN   6MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAX₃₀FEX₃₃X₃₄IAN₃₇ C2FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN   7MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALRKEAAAIRDELQAYRHN   8MGSWAEFKQRLAAIKTRLEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN   9MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAAFEKEIAAFESELQAYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN  10MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN  11MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN  12MGSWVEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYK CD137 F3GKGNPEVEKLRQRAAFIRFRLQAYRHN  13MGSWVEFANRLWAIDQRLFALGGSEAELAAFEKEIAAFESELQAYKG CD137 F3KGNPEVEHLRDQAAFIRHKLQAYRHN  14MGSWYEFRHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKG CD137 F3KGNPEVEGLREAAAFIRAKLQAYRHN  15MGSWYEFSMRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYK CD137 F3GKGNPEVEALRAKAAYIRWKLQAYRHN  16MGSWFEFNHRLWAINERLYALGGSEAELAAFEKEIAAFESELQAYKG CD137 F3KGNPEVERLRSMAAFIRYKLQAYRHN  17MGSWYEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYK CD137 F3GKGNPEVEYLRETAAHIRTRLQAYRHN  18MGSWYEFHYRLHAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKG CD137 F3KGNPEVEELRIKAAFIRDRLQAYRHN  19MGSWAEFKQRLAAIKTRLEALGGSEAELAAFLGEIWAFEMELAAYK CD137 F2GKGNPEVEALGREAAAIRMELQAYRHN  20MGSWYEFDLRLHAIYDRLVALGGSEAELAAFEKEIAAFESELQAYKG CD47 F3KGNPEVEILRDNAAYIRQMLQAYRHN  21MGSWTEFTYRLSAIEWRLWALGGSEAELAWFEQKIAFFEDFLQYYK CD47 C2GKGNPEVEALKHEAGAILNELMAYRHN  22MGSWAEFDHRLHAIRERLHALGGSEAELAAFEKEIAAFESELQAYKG CD47 F3KGNPEVEILRGNAAYIRALLQAYRHN  23MGSWTEFVGRLAAIEFRLWALGGSEAELAWFEAHIAFFEDYLQWYK CD47 C2GKGNPEVEALREEAGAIMEELKAYRHN  24MGSWTEFYSRLEAIWVRLQALGGSEAELAMFEDRIAHFEWFLQQYK CD47 C2GKGNPEVEALHEEAIAIRKELAAYRHN  25MGSWHEFHDRLQAIHERLYALGGSEAELAAFEKEIAAFESELQAYKG CTLA4 F3KGNPEVESLRIAAAHIRQVLQAYRHN  26MGSWNYFKDHLAWIKNSLEALGGSEAELAHFETAIASFERQLQEYKG DR5 F1KGNPEVEALRKEAAAIRDELQAYRHN  27MGSWLYFKEHLAHIKAWLEALGGSEAELAHFELAIADFEYHLQEYK DR5 F1GKGNPEVEALRKEAAAIRDELQAYRHN  28MGSWVYFKEHLAWIKTELEALGGSEAELAHFEHSIADFEMSLQFYKG DR5 F1KGNPEVEALRKEAAAIRDELQAYRHN  29MGSWFYFKQHLAWIKSYLEALGGSEAELAHFERAIAAFEQHLQMYK DR5 F1GKGNPEVEALRKEAAAIRDELQAYRHN  30MGSWHYFKDHLAEIKGLLEALGGSEAELAHFEMAIADFEHNLQYYK DR5 F1GKGNPEVEALRKEAAAIRDELQAYRHN  31MGSWHYFKGHLAEIKNHLEALGGSEAELAHFERAIAAFERSLQWYK DR5 F1GKGNPEVEALRKEAAAIRDELQAYRHN  32MGSWIYFKEHLAYIKKELEALGGSEAELAHFESAIAVFESTLQYYKGK DR5 F1GNPEVEALRKEAAAIRDELQAYRHN  33MGSWTYFKEHLAEIKYMLEALGGSEAELAHFEVAIADFEKMLQYYK DR5 F1GKGNPEVEALRKEAAAIRDELQAYRHN  34MGSWWLFKDHLAEIKTALEALGGSEAELAHFEMAIAAFEKQLQYYK DR5 F1GKGNPEVEALRKEAAAIRDELQAYRHN  35MGSWSEFYNRLDAIESRLLALGGSEAELALFEIQIARFEKVLQAYKGK KIR C2GNPEVEALRGEARAIFAELYAYRHN  36MGSWYEFYNRLYAIEIRLYALGGSEAELAAFEKEIAAFESELQAYKG KIR F3KGNPEVERLRVRAAKIRVILQAYRHN  37MGSWLWFKIFLAEIKYFLEALGGSEAELAAFDFEIHAFHVELFAYKGK KIR C1GNPEVEVLREVAAEIRWDLQAYRHN  38MGSWTEFQSRLDAIHSRLRALGGSEAELAAFEKEIAAFESELQAYKG PD-L1 F3KGNPEVELLRDDAAFIRHFLQAYRHN  39MGSWQEFDDRLNAIKARLQALGGSEAELAAFEKEIAAFESELQAYKG PD-L1 F3KGNPEVEDLRDDAAFIRRFLQAYRHN  40MGSWYEFQNRLHAIHERLNALGGSEAELAAFEKEIAAFESELQAYKG PD-L1 F3KGNPEVELLRDDAAFIRHFLQAYRHN  41MGSWFEFQDRLTAINERLSALGGSEAELAAFEKEIAAFESELQAYKGK PD-L1 F3GNPEVETLRSDAAFIRRFLQAYRHN  42MGSWYEFESRLDAIHERLHALGGSEAELAAFEKEIAAFESELQAYKG PD-L1 F3KGNPEVENLRGDAAFIRHFLQAYRHN  43MGSWYEFNHRLDAISKRLNALGGSEAELAAFEKEIAAFESELQAYKG PD-L1 F3KGNPEVEELRGDAAFIRHFLQAYRHN  44MGSWFEFENRLHAIVHRLGALGGSEAELAAFEKEIAAFESELQAYKG PD-L1 F3KGNPEVETLRADAAFIRHYLQAYRHN  45MGSWVVFKVDLATIKYILEALGGSEAELAEFEGEIAGFEYSLQFYKGK TIM3 F1GNPEVEALRKEAAAIRDELQAYRHN  46MGSWTIFKEWLAFIKTDLEALGGSEAELAFFEGWIASFEMELQKYKG PD1 F1KGNPEVEALRKEAAAIRDELQAYRHN  47MGSWVMFKWLLADIKSHLEALGGSEAELAFFEGFIAAFETHLQVYKG PD1 F1KGNPEVEALRKEAAAIRDELQAYRHN  48MGSWYAFKDYLADIKGWLEALGGSEAELAFFEIFIARFELELQAYKG PD1 F1KGNPEVEALRKEAAAIRDELQAYRHN  49MGSWAEFKQRLAAIKTRLQALGGSEAELAAFEKEIAAFESELQAYKG NoneKGNPEVEALRKEAAAIRDELQAYRHN  51MGSWVEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYK CD137 F3GKGNPEVEKLRQRAAFIRFRLQAYRHNGGGGSHHHHHH  52MGSWVEFANRLWAIDQRLFALGGSEAELAAFEKEIAAFESELQAYKG CD137 F3KGNPEVEHLRDQAAFIRHKLQAYRHNGGGGSHHHHHH  53MGSWYEFRHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKG CD137 F3KGNPEVEGLREAAAFIRAKLQAYRHNGGGGSHHHHHH  54MGSWYEFSMRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYK CD137 F3GKGNPEVEALRAKAAYIRWKLQAYRHNGGGGSHHHHHH  55MGSWFEFNHRLWAINERLYALGGSEAELAAFEKEIAAFESELQAYKG CD137 F3KGNPEVERLRSMAAFIRYKLQAYRHNGGGGSHHHHHH  56MGSWYEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYK CD137 F3GKGNPEVEYLRETAAHIRTRLQAYRHNGGGGSHHHHHH  57MGSWYEFHYRLHAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKG CD137 F3KGNPEVEELRIKAAFIRDRLQAYRHNGGGGSHHHHHH  58MGSWAEFKQRLAAIKTRLEALGGSEAELAAFLGEIWAFEMELAAYK CD137 F2GKGNPEVEALGREAAAIRMELQAYRHNGGGGSHHHHHH  60MGSWIEFEDRLDAITDRLWALGGSEAELAEFEHQIAFFEEDLQWYKG CD123 C2KGNPEVEALHMEAEAIMEELGAYRHN  61MGSWVEFEYRLDAISDRLWALGGSEAELAFFENEIASFESDLQFYKG CD123 C2KGNPEVEALMFEAEAIDDELHAYRHN  62MGSWYEFEDRLAAIEARLWALGGSEAELADFEEEIAYFEHGLQWYK CD123 C2GKGNPEVEALESEAMAIIDELHAYRHN  63MGSWYEFEERLDAIEDRLIALGGSEAELAIFEDIIAFFEQDLQYYKGKG CD123 C2NPEVEALEMEAEAISIELDAYRHN  64MGSWWEFEDRLWAIDRRLMALGGSEAELAVFEQMIAHFEQILQVYK CD123 C2GKGNPEVEALHFEAHAIGMELAAYRHN  65MGSWEEFHERLDAIDERLEALGGSEAELAFFEDDIASFEDWLQWYKG CD123 C2KGNPEVEALSREADAINFELEAYRHN  66MGSWEEFDKRLDAITRRLMALGGSEAELAEFESTIAWFEWDLQEYK CD123 C2GKGNPEVEALDWEAYAIDYELGAYRHN  67MGSWSEFVDRLDAIFDRLWALGGSEAELAWFEDTIAHFEWNLQEYK CD123 C2GKGNPEVEALNGEADAITDELHAYRHN  68MGSWWEFTDRLDAIFDRLWALGGSEAELAAFEESIAIFEQDLQYYKG CD123 C2KGNPEVEALEYEANAIQYELEAYRHN  69MGSWWEFTDRLEAIEDRLWALGGSEAELAHFEDSIAQFEQELQWYK CD123 C2GKGNPEVEALADEADAIESELHAYRHN  70MGSWEWFKSDLASIKWELEALGGSEAELAWFEHDIAEFEEDLQWYK CD123 F1GKGNPEVEALRKEAAAIRDELQAYRHN  71MGSWDHFKNDLAWIKKHLEALGGSEAELAEFEAVIAYFELYLQGYK CD123 F1GKGNPEVEALRKEAAAIRDELQAYRHN  72MGSWEFFKEVLAEIKYDLEALGGSEAELAWFETDIAGFEIDLQVYKG CD123 F1KGNPEVEALRKEAAAIRDELQAYRHN  73MGSWYDFKEDLADIKWMLEALGGSEAELAEFENVIAYFENDLQEYK CD123 F1GKGNPEVEALRKEAAAIRDELQAYRHN  74MGSWSFFKDDLAEIKYFLEALGGSEAELAMFEQTIAEFEYDLQDYKG CD123 F1KGNPEVEALRKEAAAIRDELQAYRHN  75MGSWVTFKDELADIKDFLEALGGSEAELAFFEVDIAEFEAELQFYKG CD123 F1KGNPEVEALRKEAAAIRDELQAYRHN  76MGSWSWFKEDLADIKFELEALGGSEAELAWFELDIADFEQALQQYK CD123 F1GKGNPEVEALRKEAAAIRDELQAYRHN  77MGSWWEFKEDLAEIKWFLEALGGSEAELAWFEHDIAKFEFELQYYK CD123 F1GKGNPEVEALRKEAAAIRDELQAYRHN  78MGSWDEFKEDLAHIKTDLEALGGSEAELALFEDEIADFEMYLQHYKG CD123 F1KGNPEVEALRKEAAAIRDELQAYRHN  79MGSWFMFKEELADIKDWLEALGGSEAELASFESYIAWFEQDLQWYK CD123 F1GKGNPEVEALRKEAAAIRDELQAYRHN  80MGSWQIFKGELAYIKQYLEALGGSEAELAFFEFDIAEFEEDLQYYKGK CD123 F1GNPEVEALRKEAAAIRDELQAYRHN  81MGSWYIFKEDLAEIKEELEALGGSEAELAYFEEEIALFEMELQWYKG CD123 F1KGNPEVEALRKEAAAIRDELQAYRHN  82MGSWYYFKDELADIKWDLEALGGSEAELAWFEMLIAQFELDLQWY CD123 F1KGKGNPEVEALRKEAAAIRDELQAYRHN  83MGSWFNFKEELAVIKFQLEALGGSEAELAFFEWVIADFEDDLQEYKG CD123 F1KGNPEVEALRKEAAAIRDELQAYRHN  84MGSWYMFKEELADIKWYLEALGGSEAELAWFEDDIAGFEWDLQAY CD123 F1KGKGNPEVEALRKEAAAIRDELQAYRHN  85MGSWHVFKTELADIKFYLEALGGSEAELAMFELWIAEFEHELQDYKG CD123 F1KGNPEVEALRKEAAAIRDELQAYRHN  86MGSWYVFKDELAEIKQFLEALGGSEAELAWFEDDIAEFETQLQHYKG CD123 F1KGNPEVEALRKEAAAIRDELQAYRHN  87MGSWTEFKGELAEIKWILEALGGSEAELAFFEDEIAAFEWDLQKYKG CD123 F1KGNPEVEALRKEAAAIRDELQAYRHN  88MGSWFWFKEDLAFIKEDLEALGGSEAELAWFEDGIAFFEWDLQDYK CD123 F1GKGNPEVEALRKEAAAIRDELQAYRHN  89MGSWSWFKEDLASIKAVLEALGGSEAELAFFESDIAEFEQELQYYKG CD123 F1KGNPEVEALRKEAAAIRDELQAYRHN  90MGSWILFKDDLAWIKETLEALGGSEAELAFFEDNIADFEEQLQGYKG CD123 F1KGNPEVEALRKEAAAIRDELQAYRHN  91MGSWQWFKDDLAYIKETLEALGGSEAELALFEDMIADFEFELQWYK CD123 F1GKGNPEVEALRKEAAAIRDELQAYRHN  92MGSWEEFHSRLDAIDDRLWALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEWLRWEAATIRETLQAYRHN  93MGSWSEFWQRLEAIEDRLWALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEWLRENAAMIRDELQAYRHN  94MGSWTEFAWRLDAIYDRLLALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEWLRHVAANIRRELQAYRHN  95MGSWDEFYYRLEAIEMRLGALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEELRHYAAQIRHMLQAYRHN  96MGSWIEFNMRLDAIYERLVALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEWLRKVAANIRLELQAYRHN  97MGSWSEFNMRLDAIYERLTALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEWLRHSAARIRLELQAYRHN  98MGSWVEFNIRLDAIYERLYALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEKLRHWAASIRRELQAYRHN  99MGSWDEFGRRLYAIEWRLYALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEKLREIAAVIRSNLQAYRHN 100MGSWIEFYDRLEAIYDRLDALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEWLREDAAFIRSWLQAYRHN 101MGSWTEFDRRLDAIWDRLFALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEWLREEAADIRDYLQAYRHN 102MGSWTEFDRRLDAIWDRLFALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEWLREEAADIRDYLQAYRHN 103MGSWIEFEVRLDAIYNRLAALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVERLRRYAANIRHELQAYRHN 104MGSWTEFHDRLEAIDDRLWALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEYLREEAAQIRWELQAYRHN 105MGSWYEFHHRLDAIYERLLALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEWLRSSAANIRKELQAYRHN 106MGSWHEFDQRLWAIEERLWALGGSEAELAAFEKEIAAFESELQAYK CD123 F3GKGNPEVETLRLYAALIRHDLQAYRHN 107MGSWIEFESRLWAIEDRLLALGGSEAELAAFEKEIAAFESELQAYKGK CD123 F3GNPEVEFLRLEAADIREDLQAYRHN 108MGSWYEFENREGAIGDRLWALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEWLRDEAAYIRAVLQAYRHN 109MGSWNEFYDRLSAIYFRLQALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEHLRWYAADIRMILQAYRHN 110MGSWYEFEYRLEAIEDRLWALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEYLREEAAWIRVWLQAYRHN 111MGSWVEFENRLEAIENRLWALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEWLREDAAQIRMMLQAYRHN 112MGSWYEFWDRLEAIDDRLWALGGSEAELAAFEKEIAAFESELQAYK CD123 F3GKGNPEVEALRQEAAWIREELQAYRHN 113MGSWFEFWDRLDAIEDRLYALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEELRDEAAWIRGTLQAYRHN 114MGSWTEFDRRLDAIWDRLFALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEWLREEAADIRDYLQAYRHN 115MGSWWEFEMRLEAIEDRLFALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVESLRWEAAFIRDILQAYRHN 116MGSWVEFYDRLHAIYFRLLALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEDERWYAADIRLVLQAYRHN 117MGSWYEFYNRLSAIYARLQALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEDLRWYAADIRYMLQAYRHN 118MGSWFEFWGRLEAIESRLKALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEELREHAAWIRAYLQAYRHN 119MGSWTEFSIRLEAIYDREVALGGSEAELAAFEKEIAAFESELQAYKGK CD123 F3GNPEVEVLRTYAANIRHELQAYRHN 120MGSWYEFENRLEAIEERLWALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEMLREEAAFIRDWLQAYRHN 121MGSWYEFVIRLEAIEDRLWALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEVLRWYAADIRHELQAYRHN 122MGSWIEFEDRLEAIEDRLFALGGSEAELAAFEKEIAAFESELQAYKGK CD123 F3GNPEVEWLRQEAAEIRLMLQAYRHN 123MGSWTEFNERLDAIYDREMALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEWLRASAAAIRVELQAYRHN 124MGSWSEFYLRLDAIYDRLDALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEWLRKTAANIREELQAYRHN 125MGSWSEFHVRLDAIYARLDALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVERLREWAANIRRELQAYRHN 126MGSWHEFGVRLDAIYDRLMALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEFLRQAAANIRSELQAYRHN 127MGSWYEFSMRLDAIYDRLMALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEQLRGYAANIRNELQAYRHN 128MGSWDEFGRRLYAIEWQLYALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEKLREIAAVIRSNLQAYRHN 129MGSWDEFGRRLYAIEWRLYALGGEEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEKLREIAAVIRSNLQAYRHN 130MGSWDEFGRRLYAIEWRLYALGGSEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEKLREIAAVIRENLQAYRHN 131MGSWDEFGRRLYAIEWQLYALGGEEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEKLREIAAVIRSNLQAYRHN 132MGSWDEFGRRLYAIEWQLYALGGTEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEKLREIAAVIRSNLQAYRHN 133MGSWDEFGRRLYAIEWQLYALGGGEAELAAFEKEIAAFESELQAYK CD123 F3GKGNPEVEKLREIAAVIRSNLQAYRHN 134MGSWDEFGRRLYAIEWQLYALGGEEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEKLREIAAVIRENLQAYRHN 135MGSWDEFGRRLYAIEWQLYALGGTEAELAAFEKEIAAFESELQAYKG CD123 F3KGNPEVEKLREIAAVIRENLQAYRHN 136MGSWDEFGRRLYAIEWQLYALGGGEAELAAFEKEIAAFESELQAYK CD123 F3GKGNPEVEKLREIAAVIRENLQAYRHN 137MGSWEEFELRLNAIEERLYALGGSEAELAYFEYVIADFEGNLQRYKG CD19 C2KGNPEVEALYFEADAIFEELVAYRHN 138MGSWFEFNHRLWAIFERLMALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEYLRAMAAVIRYHLQAYRHN 139MGSWEEFDGRLFAIEQRLQALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEVLRWFAAGIRDFLQAYRHN 140MGSWAEFYHRLYAIETRLSALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEYLRHWAAWIRTYLQAYRHN 141MGSWVEFSDRLYAIEERLWALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEELRELAAIIRHSLQAYRHN 142MGSWWEFEGRLYAIEERLTALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEYLREWAAWIRQMLQAYRHN 143MGSWWEFEHRLYAIEERLVALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEYLRNWAAYIRMALQAYRHN 144MGSWWEFEARLYAIEFRLSALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEYLRSWAAYIRTSLQAYRHN 145MGSWWEFEARLWAIESRLKALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEYLRHWAAYIRVILQAYRHN 146MGSWWEFEARLYAIEFRLSALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEYLRSWAAYIRTSLQAYRHN 147MGSWEEFYHRLDAIELRLYALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEYLRWYAAEIREILQAYRHN 148MGSWYEFYERLDAIDTRLWALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEFLREYAAEIRHFLQAYRHN 149MGSWNEFFDRLDAILYRLDALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEYLRFVAADIRSWLQAYRHN 150MGSWIEFDDRLLAIMDRLWALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEDLRDVAADIRHYLQAYRHN 151MGSWYEFWERLDAITFRLYALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEDLRTWAADIRAILQAYRHN 152MGSWEEFYIRLDAIMERLWALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEDLRYAAADIRHFLQAYRHN 153MGSWIEFEERLYAIETRELALGGSEAELAAFEKEIAAFESELQAYKGK CD19 F3GNPEVEFLRVVAADIREWLQAYRHN 154MGSWIEFEHRLSAINDRLYALGGSEAELAAFEKEIAAFESELQAYKGK CD19 F3GNPEVEDLREWAADIRSLLQAYRHN 155MGSWFEFEMRLDAIMARLWALGGSEAELAAFEKEIAAFESELQAYK CD19 F3GKGNPEVEDLRYAAADIRDYLQAYRHN 156MGSWYEFVYRLDAIYDRLWALGGSEAELAAFEKEIAAFESELQAYK CD19 F3GKGNPEVEDLRYAAADIRDFLQAYRHN 157MGSWVEFEDRLDAILERLWALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEDLRELAADIRDFLQAYRHN 158MGSWFEFEERLIAIEERLFALGGSEAELAAFEKEIAAFESELQAYKGK CD19 F3GNPEVEYLRWIAADIRDVLQAYRHN 159MGSWIEFADRLDAILDRLDALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEWLREIAADIRAYLQAYRHN 160MGSWLEFEYRLDAILDRLFALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEDLREVAADIRMLLQAYRHN 161MGSWYEFHDRLDAITNRLYALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEDLRDWAADIRVWLQAYRHN 162MGSWQEFEQRLDAINWRLWALGGSEAELAAFEKEIAAFESELQAYK CD19 F3GKGNPEVEELREWAADIRIFLQAYRHN 163MGSWYEFYSRLDAIDSRLYALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEFLRDYAAEIRRYLQAYRHN 164MGSWEEFHDRLEAISDRLWALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEDLRDWAADIRFYLQAYRHN 165MGSWWEFDERLYAIEDRLFALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEWLRIVAADIREILQAYRHN 166MGSWEEFEYRLMAIEVRLWALGGSEAELAAFEKEIAAFESELQAYKG CD19 F3KGNPEVEVLREIAADIRQILQAYRHN 167MGSWVVFKQRLAYIKDLLEALGGSEAELAYFEMSIAFFEEDLQVYKG CD22 F1KGNPEVEALRKEAAAIRDELQAYRHN 168MGSWYEFKNDLAWIKVHLEALGGSEAELAYFEFRIAHFENALQYYK CD22 F1GKGNPEVEALRKEAAAIRDELQAYRHN 169MGSWVEFYNRLWAIDHRLHALGGSEAELAAFEKEIAAFESELQAYK CD22 F3GKGNPEVEVLRYHAASIRVTLQAYRHN 170MGSWSEFYDRLHAIHHRLYALGGSEAELAAFEKEIAAFESELQAYKG CD22 F3KGNPEVEALRDTAAFIRTRLQAYRHN 171MGSWKEFHFRLHAIEHRLIALGGSEAELAAFEKEIAAFESELQAYKGK CD22 F3GNPEVEFLRAKAANIRTHLQAYRHN 172MGSWFEFHGRLHAIYGRLSALGGSEAELAAFEKEIAAFESELQAYKG CD22 F3KGNPEVEHLRAHAAHIRDHLQAYRHN 173MGSWYEFADRLHAIHQRLYALGGSEAELAAFEKEIAAFESELQAYKG CD22 F3KGNPEVEALRMTAAFIRSRLQAYRHN 174MGSWNEFYNRLHAIHQRLYALGGSEAELAAFEKEIAAFESELQAYKG CD22 F3KGNPEVESLRQTAAYIRDRLQAYRHN 175MGSWNEFADRLHAIHQRLYALGGSEAELAAFEKEIAAFESELQAYKG CD22 F3KGNPEVESLRMTAAFIRSRLQAYRHN 176MGSWTEFSYRLGAIQSRLHALGGSEAELAAFEKEIAAFESELQAYKG CD22 F3KGNPEVEHLRYNAAKIRHFLQAYRHN 177MGSWQEFTTRLEAIYHRLRALGGSEAELANFEGFIAEFEGNLQMYKG DR5 C2KGNPEVEALVHEAYAIMEELHAYRHN 178MGSWVEFFDRLKAIHDRLEALGGSEAELAHFEKLIAHFEHRLQNYKG DR5 C2KGNPEVEALEKEADAILYELAAYRHN 179MGSWYYFKHHLAWIKMELEALGGSEAELAHFESSIASFERDLQQYK DR5 F1GKGNPEVEALRKEAAAIRDELQAYRHN 180MGSWVEFHIRLHAIQYRLYALGGSEAELAAFEKEIAAFESELQAYKG DR5 F3KGNPEVEELRHWAAFIRLQLQAYRHN 181MGSWNEFHDRLNAIHARLHALGGSEAELAAFEKEIAAFESELQAYKG PD-L1 F3KGNPEVENLRDDAAFIRRFLQAYRHN 182MGSWYEFTVRLEAIHERLKALGGSEAELAAFEKEIAAFESELQAYKG PD-L1 F3KGNPEVEILRDDAAFIRRFLQAYRHN 183MGSWKEFDDRLNAIKARLQALGGSEAELAAFEKEIAAFESELQAYKG PD-L1 F3KGNPEVEDLRDDAAFIRRFLQAYRHN 184MGSWYEFDDRENAIHDRLQALGGSEAELAAFEKEIAAFESELQAYKG PD-L1 F3KGNPEVEDLRDDAAFIRRFLQAYRHN 185MGSWNEFKNRLDAIHKRLNALGGSEAELAAFEKEIAAFESELQAYKG PD-L1 F3KGNPEVENLRDDAAFIRHFLQAYRHN 186MGSWTEFEQRLEAIHNRLQALGGSEAELAAFEKEIAAFESELQAYKG PD-L1 F3KGNPEVEELRNDAAFIRHFLQAYRHN

In some embodiments, the invention comprises one or more the sequencesidentified on Table 1. In other embodiments, the invention comprises oneor more the sequences with 60-70%, 70-75%, 75-80%, 80-85%, 85-90%,95-99% homology (and overlapping ranges therein) with those sequencesidentified on Table 1. In several embodiments, the sequences having suchhomology are functionally similar or identical as compared to therespective sequence identified on Table 1. In several embodiments, theinvention comprises one or more polypeptides that compete with (whollyor partially) one or more of the sequences in Table 1 for its respectivetarget. In several embodiments, the competition can be assessed by astandard competition assay. In some embodiments, competition does notrequire that the competing polypeptide compete for the same specifictarget as those polypeptides of Table 1, rather they can compete bybinding a sterically inhibiting epitope, an overlapping epitope, etc.

Affinity Maturation of DBDpp

Affinity maturation strategies can be used to generate high affinityDBDpp that can be used in the DBDpp fusion proteins described herein.

Mutagenesis libraries may be created and screened to further optimizethe sequence of the best binders. Lowman, Ann. Rev. Biophys. Biomol.Struct. 26: 401-24 (1997).

An improved DBDpp that specifically binds a desired target can also beprepared based on a known DBDpp sequence. For example, at least one,two, three, four, five, or more amino acid mutations (e.g., conservativeor non-conservative substitutions), deletions or insertions can beintroduced into a known DBDpp sequence and the resulting DBDpp can bescreened for binding to the desired target and biological activity, suchas the ability to antagonize target biological activity or agonizetarget biological activity.

Articles of Manufacture

Articles of manufacture, including, kits, are provided herein. Thearticle of manufacture may comprise a container and a label or packageinsert on or associated with the container. Suitable containers include,for example, bottles, vials or syringes. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds one or more DBDpp, nucleic acids encoding DBDpp and/or vectors orhost cells of the present disclosure. The label or package insert mayinclude directions for performing affinity based screening, detection,and/or purification.

Also provided are kits containing a DBDpp. Such kits have usesincluding, but not limited to detecting or isolating the target ofinterest to which the DBDpp specifically binds. Such assay kit may beuseful in screening for the presence of a target of interest and/orquantitating the concentrations of a target of interest in a fluid, suchas, a biological fluid (e.g., blood, serum, or synovial fluid).

In one embodiment a DBDpp assay kit is contemplated which comprises oneor more containers of a DBDpp that specifically binds a target ofinterest and, optionally, a detection means for determining the presenceor absence of a target/DBDpp interaction or the absence thereof. The kitfurther optionally contains target of interest protein that may be used,for example as a control or standard. The DBDpp may be free or expressedon the surface of a host cell or on the surface of a bacteriophage. In aspecific embodiment, the DBDpp or target of interest provided in the kitis labeled. Any label known in the art can be used. In some embodiments,the label is selected from the group consisting of biotin, a fluorogen,an enzyme, an epitope, a chromogen, or a radionuclide. In someembodiments, the DBDpp is immobilized on a solid support. The detectionmeans employed to detect the label will depend on the nature of thelabel and can be any known in the art, e.g., film to detect aradionuclide; an enzyme substrate that gives rise to or amplifies adetectable signal to detect the presence of a target of interest.

Preferably, the kit further comprises a solid support for the DBDpp,which may be provided as a separate element or on which a DBDpp thatspecifically binds a target of interest is immobilized. Hence, the DBDppthat specifically binds the target of interest in the kit may beimmobilized on a solid support, or they may be immobilized on suchsupport that is included with the kit or provided separately from thekit. Preferably, DBDpp is coated on a microtiter plate. In someembodiments, the detection involves a signal amplifying molecule. Wherethe signal amplifying molecule is an enzyme, the kit optionally furtherincludes substrates and cofactors required by the enzyme, and where theamplifying molecule is a fluorophore. The kit optionally furtherincludes a dye precursor that provides the detectable chromophore.

The kit may also contain instructions for carrying out the assay as wellas other additives such as stabilizers, washing and incubation buffers,and the like. The components of the kit will be provided inpredetermined ratios, with the relative amounts of the various reagentssuitably varied to provide for concentrations in solution of thereagents that substantially maximize the sensitivity of the assay and/orthe ability to purify the target of interest. Particularly, the reagentscan be provided as dry powders, usually lyophilized, includingexcipients, which on dissolution will provide for a reagent solutionhaving the appropriate concentration for combining with the sample to betested.

Various formats and techniques for binding assays that can be used areknown in the art and include but are not limited to, immobilization tofilters such as nylon or nitrocellulose; two-dimensional arrays, enzymelinked immunosorbent assay (ELISA), radioimmuno-assay (RIA), competitivebinding assays, direct and indirect sandwich assays, immunoprecipitationassays, fluorimetric microvolume assay technology (FMAT™), Luminex™system assays, fluorescent resonance energy transfer (FRET),bioluminescence resonance energy transfer (BRET), electroimmunoassays,AlphaScreen™, nanoparticle-derived techniques, and surface plasmonresonance (SPR).

Binding assays can be homogeneous or semi-homogeneous. A homogeneousassay is an assay where all the components are mixed together,incubated, and then analyzed. A semi-homogeneous assay is one where themajority of the reaction takes place as a complex mixture, but a washingstep is required prior to the addition of a final reagent and analysis,in contrast to a typical stepwise assembly sandwich assay where eachcomponent is added then washed off before the next component is added.In some embodiments the assay is an immunoassay. In certain embodimentsthe assay is a semi-homogeneous Enzyme Immuno-Assay (EIA),

Applications

DBDpp, whether alone, as fusion proteins, as chemical conjugates or asother embodiments described herein, have a variety of applications. Insome embodiments, DBDpp are used as detection reagents, capturereagents, separation reagents, diagnostic reagents or analyticalreagents. Some embodiments have in vivo, in vitro and/or ex vivoapplications. Methods that employ the DBDpp in vitro can be performed indifferent formats, such as in microtiter plates, in protein arrays, onbiosensor surfaces, on tissue sections, and in additional formats thatwould be apparent to a person skilled in the art. Likewise, methods thatemploy the DBDpp in vivo can be used in different formats that includebut are not limited to DBDpp-Fc fusion proteins, CAR cells, and DBDppmulti-specific antibodies. In particular embodiments DBDpp such as DBDppfusion proteins are used as a therapeutic agent.

Analytical and Diagnostic Applications

Whether alone, as fusion proteins, as chemical conjugates or as otherembodiments described herein, DBDpp have a variety of applications. Insome embodiments, DBDpp are used as detection reagents of targets ofinterest in a variety of different sample types.

In one embodiment a DBDpp are used to detect targets of interest insolutions involved in manufacturing processes, such as proteinexpression and purification. Samples may include, but are not limitedto, water, buffers, in-process purification samples, bulk drug substanceand final drug product. In still additional embodiments, the DBDpp canbe used to detect and/or remove impurities or contaminants from asample, such as a water supply source or water (or other fluid) used inmanufacturing.

In another embodiment, DBDpp are used to detect targets of interest indiagnostic samples. Samples may include, but are not limited to tissuehomogenates, cell extracts, biopsy samples, sera, plasma, lymph, blood,blood fractions, urine, synovial fluid, spinal fluid, saliva, mucous,sputum, pleural fluid, nipple aspirates, fluid of the respiratory,intestinal, and genitourinary tracts, tear fluid, breast milk, fluidfrom the lymphatic system, semen, cerebrospinal fluid, intra-organsystem fluid, ascitic fluid, tumor cyst fluid, amniotic fluid, and mediaor lysate from cultured cells.

In one embodiment, the DBDpp are useful for detecting the presence of afactor or multiple factors (e.g., antigens or organisms) in a biologicalsample. The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell, tissue or fluid. In certain embodiments, such tissuesinclude normal and/or cancerous tissues.

Various formats and techniques for detection are known in the art andinclude but are not limited to Western Blot analysis,Immunohistochemistry, ELISA, FACS analysis, enzymatic assays,autoradiography and any of the binding assays mentioned herein.

In one embodiment, a method is provided for detecting a target ofinterest in a solution containing the target comprising: (a) contactingthe solution with a DBDpp that specifically binds the target of interestunder conditions suitable for specific binding of the DBDpp to thetarget and (b) detecting binding of the DBDpp and target. The DBDpp maybe either free or immobilized. Sufficient time is allowed to permitbinding between the target of interest and the DBDpp, and non-bindingcomponents in the solution or mixture are removed or washed away. Theformation of a binding complex between the DBDpp and the target ofinterest can then be detected, for example, by detecting the signal froma label on the DBDpp, which is one component of the binding complex. Alabel may be any label that generates a signal that can be detected bystandard methods, such as a fluorescent label, a radioactive compound,or an enzyme that reacts with a substrate to generate a detectablesignal. Examples of suitable labels for such purposes are describedherein and/or otherwise known in the art.

DBDpp that bind to a of interest can be detectably labeled through theuse of radioisotopes, affinity labels (such as biotin, avidin, etc.),enzymatic labels (such as horseradish peroxidase, alkaline phosphatase,etc.) using methods known in the art, such as described in WO 00/70023and (Harlow and Lane (1989) Antibodies, Cold Spring Harbor Laboratory,pp. 1-726).

The detectable marker or label can be any which is capable of producing,either directly or indirectly, a measurable signal, such as aradioactive, chromogenic, luminescence, or fluorescent signal, which canbe used to quantitate the amount of bound detectable moiety or label ina sample. Detectable labels known in the art include radioisotopes, suchas 3H, 14C, 32P, 35S, or 125I, electrochemiluminescent labels (such asRuthenium (Ru)-based catalyst in conjunction with substrates, etc.),luminescent or bioluminescent labels (e.g., Europium, Vanadium),fluorescent or chemiluminescent compounds, such as fluoresceinisothiocyanate, rhodamine, or luciferin, enzymes (e.g., enzyme, such asalkaline phosphatase, beta-galactosidase, or horseradish peroxidase),colorimetric labels such as colloidal gold, colored glass or plasticbeads (e.g., polystyrene, polypropylene, latex, etc.), paramagneticatoms or magnetic agents, electron-dense reagents, a nano- or micro-beadcontaining a fluorescent dye, nanocrystals, a quantum dot, a quantumbead, a nanotag, dendrimers with a fluorescent label, amicro-transponder, an electron donor molecule or molecular structure, ora light reflecting particle, the microparticles may be nanocrystals orquantum dots. Nanocrystals are substances that absorb photons of light,then re-emit photons at a different wavelength (fluorophores). Inaddition, additional fluorescent labels, or secondary antibodies may beconjugated to the nanocrystals. Nanocrystals are commercially availablefrom sources such as Invitrogen and Evident Technologies (Troy, N.Y.).Other labels include E)-5-[2-(methoxycarbonyl) ethenyl]cytidine, whichis a nonfluorescent molecule that when subjected to ultraviolet (UV)irradiation yields a product, 3beta-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine, which displays astrong fluorescence signal.

Competitive inhibition can be determined by any method known in the art,for example, competition ELISA assays. A DBDpp, such as a DBDpp fusionprotein (e.g., a DBDpp-Fc, DBDpp-CAR, a DBDpp-scFv), or other moleculeis said to “competitively inhibit” binding of a reference molecule to agiven epitope if it binds to that epitope to the extent that it blocks,to some degree, binding of the reference molecule to the epitope. Asused herein, a DBDpp (e.g., a DBDpp fusion protein), or other moleculecan be said to competitively inhibit binding of the reference moleculeto a given epitope, for example, by at least 90%, at least 80%, at least70%, at least 60%, at least 50%, by at least 40%, at least 30%, or atleast 20%. The terms “compete,” “ability to compete” and “competes with”are relative terms used to describe a DBDpp, such as a DBDpp fusionprotein, that produce at least 20%, at least 30%, at least 40%, or atleast 50% inhibition of binding of a reference molecule to a target by aDBDpp such as a DBDpp fusion protein (e.g., a DBDpp-Fc, DBDpp CAR, aDBDpp-scFv, and an antibody-comprising a DBDpp) as determined in astandard competition assay as described herein or otherwise known in theart, including, but not limited to, competitive assay systems usingtechniques such as radioimmunoassays (RIA), enzyme immunoassays (EIA),preferably the enzyme linked immunosorbent assay (ELISA), “sandwich”immunoassays, immunoradiometric assays, fluorescent immunoassays,luminescent, electrochemical luminescent, and immunoelectrophoresisassays. Methods for determining binding and affinity of candidatebinding molecules are known in the art and include, but are not limitedto, affinity chromatography, size exclusion chromatography, equilibriumdialysis, fluorescent probe displacement, and plasma resonance.

Affinity Purification

In purification based on affinity chromatography, target proteins areselectively isolated according to their ability to specifically andreversibly bind to a ligand that has typically been covalently coupledto a chromatographic matrix. In one embodiment, DBDpp can be used asreagents for affinity purification of targets of interest from eitherrecombinant sources or natural sources such as biological samples (e.g.,serum).

In another embodiment, a method for isolating a target of interest froma solution that contains the target of interest is provided. Such methodcomprises: (a) contacting the solution with a DBDpp under conditionsthat permit binding of the DBDpp to the target of interest; and (b)recovering the target of interest. In another embodiment, a method isprovided for isolating a target of interest from a solution thatcontains the target of interest, comprising: (a) contacting the solutionwith a DBDpp under conditions suitable for specific binding of the DBDppto the target; and (b) separating the complex(es) formed by the targetof interest and/or DBDpp from other components of the solution. In afurther embodiment, the method further comprises the steps of: (c)dissociating the DBDpp from the target of interest, and (d) recoveringthe dissociated target of interest.

In some embodiments, the DBDpp that specifically binds a target ofinterest is immobilized on beads and then used to affinity purify thetarget protein.

Methods of covalently coupling proteins to a surface are known by thoseof skill in the art, and peptide tags that can be used to attach DBDppto a solid surface are known to those of skill in the art. Further,DBDpp can be attached (i.e., coupled, linked, or adhered) to a solidsurface using any reagents or techniques known in the art. In someembodiments, the solid support is selected from: beads, glass, slides,chips and gelatin. Thus, a series of DBDpp can be used to make an arrayon a solid surface using techniques known in the art. For example, U.S.Publ. No. 2004/0009530 discloses methods for preparing arrays. Thecontents of U.S. Publ. No. 2004/0009530 are herein incorporated byreference in its entirety.

In another embodiment, a DBDpp is used to isolate a target of interestby affinity chromatography. Any conventional method of chromatographymay be employed. In some embodiments, a DBDpp is immobilized on a solidsupport. The DBDpp can be immobilized on the solid support usingtechniques and reagents described herein or otherwise known in the art.Suitable solid supports are described herein or otherwise known in theart and in specific embodiments are suitable for packing achromatography column. The immobilized DBDpp can then be loaded orcontacted with a solution under conditions favorable to form a complexbetween the DBDpp and the target of interest. Non-binding materials canbe washed away. Suitable wash conditions can readily be determined byone of skill in the art. Examples of suitable wash conditions includebut are not limited to PBS/0.01% Tween 20, pH7.2 and 1M NaCl/10 mM Tris,pH7.5. Tris wash buffers may be preferable since phosphates canprecipitate in 50% ethylene glycol. In general, non-limiting terms, washbuffers are pH7.0, optionally containing 0.0 to 1.5 M NaCl, morepreferably 1M NaCl. Additionally, wash buffers may optionally contain amild detergent, such as, Tween 20, Tween 80, or NP-80. The target ofinterest can be eluted from the DBDpp binding complex by introducingsolution conditions that favor dissociation of the binding complex.Suitable elution solutions can readily be determined by one of skill inthe art and include but are not limited to 50% ethylene glycol/10 mMNaOAc. By way of non-limiting example, useful elution buffers, contain40-60% ethylene glycol, preferably 50% ethylene glycol; and 50-100 mMNaOAc with a pH in the range of pH 4-7, more preferably, pH 4-6 and mostpreferably pH 4.5-5.5. Preferably, a fast flow affinity chromatographictechnique is used to bind the DBDpp to the target of interest and fromwhich the purified target of interest is eluted.

Alternatively, chromatography can be carried out by mixing a solutioncontaining the target of interest and the DBDpp, then isolatingcomplexes of the target of interest and DBDpp. For this type ofseparation, many methods are known and can routinely be applied. Forexample, the DBDpp may be immobilized on a solid support such as beads,then separated from a solution along with the target of interest byfiltration. In another example, the DBDpp may be a fusion protein thatcontains a peptide tag, such as a poly-HIS tail or streptavidin bindingregion, which can be used to isolate the DBDpp after complexes haveformed using an immobilized metal affinity chromatographic resin orsteptavidin-coated substrate. Once separated, the target of interest canbe released from the DBDpp under elution conditions and recovered in apurified form.

Therapeutics

The DBD described herein are useful in a variety of applicationsincluding, but not limited to, therapeutic treatment methods, which maybe in vitro, ex vivo, or in vivo methods.

The application as a therapeutic entity is an attribute of the targetbinding specificity of the DBDpp. The incorporation of DBDpp withinvarious molecular compositions, (e.g., a DBD-antibody fusions, DBD-drugconjugates and DBD-chimeric receptors) affords application in a varietyof therapeutic indications and modalities, which include, but notlimited to soluble and cell-associated compositions.

In one embodiment, the DBDpp is a soluble fusion protein (schematicallyshown in FIG. 5C and made up of an optional epitope tag 10 and atargeting domain 20) that binds to a target that is associated with adisease or disorder of the metabolic, cardiovascular, musculoskeletal,neurological, or skeletal system. In other embodiments, the DBDpp is asoluble fusion protein that binds to a target that is associated withyeast, fungal, viral or bacterial infection or disease. In someembodiments, the DBDpp is a soluble fusion protein that binds to atarget that is associated with a disease or disorder of the immunesystem.

Also provided are therapeutic compositions useful for practicingtherapeutic methods described herein. In one embodiment, therapeuticcompositions provided herein contain a physiologically tolerable carriertogether with at least one species of DBDpp fusion as described herein,dissolved or dispersed therein as an active ingredient. In anotherembodiment, therapeutic compositions provided herein contain aphysiologically tolerable carrier together with at least one species ofa DBDpp as described herein, dissolved or dispersed therein as an activeingredient. In a preferred embodiment, therapeutic composition is notimmunogenic when administered to a human patient for therapeuticpurposes.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in theart. Typically such compositions are prepared as sterile injectableseither as liquid solutions or suspensions, aqueous or non-aqueous.However, solid forms suitable for solution, or suspensions, in liquidprior to use can also be prepared. The preparation can also beemulsified. Thus, a DBDpp-containing composition can take the form ofsolutions, suspensions, tablets, capsules, sustained releaseformulations or powders, or other compositional forms. In someembodiments, the DBDpp compositions (e.g., a DBDpp fusion proteins) areformulated to ensure or optimize distribution in vivo, For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds andif so desired, the compositions are prepared so as to increase transferacross the BBB, by for example, formulation in liposomes. For methods ofmanufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811, 5,374,548,and 5,399,331. The liposomes can comprise one or more moieties that areselectively transported into specific cells or organs, thus enhancetargeted drug delivery (see, e.g., Ranade, Clin. Pharmacol. 29:685(1989)).

The DBDpp (e.g. DBDpp fusion protein) can be mixed other activeingredients and/or excipients that are pharmaceutically acceptable andcompatible with the active ingredient and in amounts suitable for use intherapeutic methods described herein. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents and the like which enhance theeffectiveness of the active ingredient.

Therapeutic DBDpp can include pharmaceutically acceptable salts of thecomponents therein. Pharmaceutically acceptable salts include the acidaddition salts (formed with the free amino groups of the polypeptide)that are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, tartaric, mandelicand the like. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium or ferric hydroxides, and such organic bases asisopropylamine, trimethylarnine, 2-ethylamino ethanol, histidine,procaine and the like.

Physiologically tolerable carriers are known in the art. Exemplary ofliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH value, physiological salineor both, such as phosphate-buffered saline. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, propylene glycol, polyethyleneglycol, and other solutes.

Liquid compositions can also contain liquid phases in addition to, andto the exclusion of water. Exemplary of such additional liquid phasesare glycerin, vegetable oils such as cottonseed oil, organic esters suchas ethyl oleate, and water-oil emulsions.

In one embodiment, a therapeutic composition contains a DBDpp fusionprotein, typically in an amount of at least 0.1 weight percent of DBDppfusion protein per weight of total therapeutic composition. A weightpercent is a ratio by weight of DBDpp fusion per total composition.Thus, for example, 0.1 weight percent is 0.1 grams of DBDpp per 100grams of total composition.

A DBDpp fusion protein-containing therapeutic composition typicallycontains about 10 micrograms (μg) per milliliter (ml) to about 100milligrams (mg) per ml of DBDpp fusion protein as active ingredient pervolume of composition, and more preferably contains about 1 mg/ml toabout 10 mg/ml (i.e., about 0.1 to 1 weight percent).

The dosage ranges for the administration of the DBDpp (e.g., a DBDppfusion protein) are those large enough to produce the desired effect inwhich the disease symptoms mediated by the target molecule areameliorated. The dosage should not be so large as to cause adverse sideeffects, such as hyperviscosity syndromes, pulmonary edema, congestiveheart failure, and the like. Generally, the dosage will vary with theage, condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The dosage can be adjusted by theindividual physician in the event of any complication.

The DBDpp (e.g., a DBDpp fusion protein) can be administeredparenterally by injection or by gradual infusion over time. Although thetarget molecule can typically be accessed in the body by systemicadministration and therefore most often treated by intravenousadministration of therapeutic compositions, other tissues and deliverymeans are contemplated where there is a likelihood that the tissuetargeted contains the target molecule. Thus, DBDpp can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, transdermally, and can be delivered by peristaltic means.DBDpp fusion proteins can also be delivered by aerosol to airways andlungs.

Therapeutic compositions containing a DBDpp can be conventionallyadministered intravenously, as by injection of a unit dose, for example.The term “unit dose” when used in reference to a therapeutic compositionprovided herein refers to physically discrete units suitable as unitarydosage for the subject, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect inassociation with the required diluent; e.g., carrier, or vehicle. In aspecific embodiment, therapeutic compositions containing a DBDpp areadministered subcutaneously.

In some embodiments, the DBDpp (e.g., a DBDpp fusion protein) isadministered in a manner compatible with the dosage formulation, and ina therapeutically effective amount. The quantity to be administereddepends on the subject to be treated, capacity of the subject's systemto utilize the active ingredient, and degree of therapeutic effectdesired. Precise amounts of active ingredient required to beadministered depend on the judgment of the practitioner and are peculiarto each individual. However, suitable dosage ranges for systemicapplication are disclosed herein and depend on the route ofadministration. Suitable regimes for administration are also variable,but are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated.

The DBDpp compositions are formulated, dosed, and administered in afashion consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Thedosage ranges for the administration of the DBDpp are those large enoughto produce the desired effect in which the disease symptoms mediated bythe target molecule are ameliorated. The dosage should not be so largeas to cause adverse side effects, such as, hyperviscosity syndromes,pulmonary edema, congestive heart failure, and the like. Generally, thedosage will vary with the age, condition, sex and extent of the diseasein the patient and can be determined by one of skill in the art. Thedosage can be adjusted by the individual physician in the event of anycomplication.

The dosage schedule and amounts effective for therapeutic andprophylactic uses, i.e., the “dosing regimen,” will depend upon avariety of factors, including the cause, stage and severity of thedisease or disorder, the health, physical status, age of the mammalbeing treated, and the site and mode of the delivery of the DBD.Therapeutic efficacy and toxicity of the complex and formation can bedetermined by standard pharmaceutical, pharmacological, andtoxicological procedures in cell cultures or experimental animals. Dataobtained from these procedures can likewise be used in formulating arange of dosages for human use. Moreover, therapeutic index (i.e., thedose therapeutically effective in 50 percent of the population dividedby the dose lethal to 50 percent of the population (ED50/LD50)) canreadily be determined using known procedures. The dosage is preferablywithin a range of concentrations that includes the ED50 with little orno toxicity, and may vary within this range depending on the dosage formemployed, sensitivity of the patient, and the route of administration.

The dosage regimen also takes into consideration pharmacokineticsparameters known in the art, such as, drug absorption rate,bioavailability, metabolism and clearance (see, e.g., Hidalgo-Aragones,J. Steroid Biochem. Mol. Biol. 58:611-617 (1996); Groning et al.,Pharmazie 51:337-341 (1996); Fotherby, Contraception 54:59-69 (1996);and Johnson et al., J. Pharm. Sci. 84:1144-1146 (1995)). It is wellwithin the state of the art for the clinician to determine the dosageregimen for each subject being treated. Moreover, single or multipleadministrations of DBDpp compositions can be administered depending onthe dosage and frequency as required and tolerated by the subject. Theduration of prophylactic and therapeutic treatment will vary dependingon the particular disease or condition being treated. Some diseases areamenable to acute treatment whereas others require long-term, chronictherapy. DBDpp can be administered serially, or simultaneously with theadditional therapeutic agent.

In some embodiments, the DBDpp is administered at about 1 mg/kg to about50 mg/kg, about 1 mg/kg to about 25 mg/kg, about 1 mg/kg to about 20mg/kg, about 1 mg/kg to about 15 mg/kg, about 1 mg/kg to about 10 mg/kg,or about 1 mg/kg to about 5 mg/kg.

In another embodiment, a DBDpp is administered in combination with moreone or more additional therapeutics.

A therapeutically effective amount of a DBDpp, such as a DBDpp fusionprotein, can be an amount such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 0.1 microgram (μg) per milliliter (ml) toabout 100 μg/ml, preferably from about 1 μg/ml to about 5 μg/ml, andusually about 5 μg/ml. Stated differently, the dosage can vary fromabout 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg toabout 200 mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg,in one or more dose administrations daily, for one or several days.

In one embodiment the disease or disorder is a disease or disorder ofthe immune system, such as inflammation or an autoimmune disease.

In some embodiments, the DBDpp is a soluble protein that specificallybinds to a target that is associated with a disease or disorder of themetabolic, cardiovascular, musculoskeletal, neurological, or skeletalsystem.

In other embodiments, the DBDpp is a soluble protein that specificallybinds to a target that is associated with yeast, fungal, viral orbacterial infection or disease. In some embodiments, the DBDpp is asoluble protein that specifically binds to a target that is associatedwith a disease or disorder of the immune system.

In one embodiments, the DBDpp fusion proteins are useful for inhibitingtumor growth, reducing neovascularization, reducing angiogenesis,inducing differentiation, reducing tumor volume, and/or reducing thetumorigenicity of a tumor.

In some embodiments, the DBDpp described herein are useful for treatingcancer. Thus, in some embodiments, the invention provides methods oftreating cancer comprise administering a therapeutically effectiveamount of a DBDpp (e.g. a DBDpp fusion) to a patient.

Cancers that can be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors. Thecancers can comprise non-solid tumors (such as hematological tumors, forexample, leukemias and lymphomas) or can comprise solid tumors. Types ofcancers to be treated with the DBDpp include, but are not limited to,carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoidmalignancies, benign and malignant tumors, and malignancies e.g.,sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatrictumors/cancers are also included.

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma,and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostatecancer, hepatocellular carcinoma, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroidcarcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervicalcancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNStumors (such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pineaioma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

In another embodiment, the DBDpp described herein are useful fortreating a patient having hematological cancers. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblasts, promyeiocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

In additional embodiments, the DBDpp fusion protein binds (1) a targeton a cell or tissue of interest (e.g., a tumor antigen on a tumor cell)and (2) a target on an effector cell, such as, a T-cell receptormolecule. According to one embodiment, the binding of one or moretargets by the DBDpp fusion protein is used to direct an immune responseto an infectious agent, cell, tissue, or other location of interest in apatient. For example, in some embodiments a DBDpp specifically binds atarget on the surface of an effector cell. Thus, in some embodiments, aDBDpp specifically binds a target on the surface of a T cell. Inspecific embodiments a DBDpp specifically binds CD3. In otherembodiments, a DBDpp specifically binds CD2. In a further embodiment, aDBDpp specifically binds the T-cell receptor (TCR). According toadditional embodiments, a DBDpp specifically binds a target on thesurface of a Natural Killer Cell. Thus, in some embodiments, a DBDppspecifically binds a NKG2D (Natural Killer Group 2D) receptor. Inadditional embodiments a DBDpp specifically binds CD16 (i.e., Fc gammaRIII) CD64 (i.e., Fc gamma RI), or CD32 (i.e., Fc gamma RII).

In one embodiment, a DBDpp fusion protein binds a target on a leukocyteand a tumor antigen on a tumor cell. In some embodiments, the DBDppfusion protein binds NKG2D. In a further embodiment, a DBDpp fusionprotein binds NKG2D and a target selected from ErbB2, EGFR, IGF1R, CD19,CD20, CD80 and EPCAM. In one embodiment, a DBDpp fusion protein bindsCD3. In particular embodiments, the DBDpp specifically binds CD3epsilon. In one embodiment, a DBDpp fusion protein binds CD4.

In one embodiment, the DBDpp fusion is bispecific and specifically bindsto two different targets expressed on the surface of two different celltypes. In one embodiment the bispecific DBDpp fusion proteinspecifically binds to a cancer cell target and an immune effector celltarget. In one embodiment the bispecific DBDpp fusion proteinspecifically binds a target expressed on a cancer cell (e.g. CD19) and atarget expressed on the surface of a T lymphocyte (e.g., CD3).

In some embodiments, DBDpp can mimic ligand binding. In certainembodiments, a DBDpp can mimic the biological activity of a ligand (anagonist DBDpp) or inhibit the bioactivity of the ligand (an antagonistDBDpp), e.g., through competitive binding. DBDpp in DBDpp fusionproteins can also affect targets in other ways, e.g., by neutralizing,blocking, stabilizing, aggregating, or crosslinking a DBDpp target.

DBDpp Drug Conjugates

In a further embodiment a DBDpp fusion protein may be linked to otherorganic or inorganic molecules or substrates through the use ofchemically conjugation. In one embodiment, DBDpp-drug conjugates areintended to facilitate the local delivery of cytotoxic agents throughthe targeting specificity of the DBDpp. This combination of targetingspecificity and cytotoxic agent, allows targeted delivery of the drug totumors, and intracellular accumulation therein, where systemicadministration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al., Lancet pages 603-05(1986); Thorpe, “Antibody Carriers Of Cytotoxic agents In CancerTherapy: A Review,” in Monoclonal Antibodies '84: Biological AndClinical Applications, A. Pinchera et al., (ed.s), pp. 475-506) (1985)).

Cytotoxic agents include chemotherapeutic agents, growth inhibitoryagents, toxins (e.g., an enzymatically active toxin of bacterial,fungal, plant, or animal origin, or fragments thereof), radioactiveisotopes (i.e., a radioconjugate), etc. Chemotherapeutic agents usefulin the generation of such immunoconjugates include, for example,methotrexate, adriamicin, doxorubicin, melphalan, mitomycin C,chlorambucil, daunorubicin or other intercalating agents.Chemotherapeutic agents useful in the generation of suchimmunoconjugates also include antitubulin drugs, such as auristatins,including monomethyl auristatin E (MMAE) and monomethyl auristatin F(MMAF). Enzymatically active toxins and fragments thereof that can beused according to the invention include diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain, ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes.

In one embodiment, a DBDpp (e.g., a DBDpp fusion protein) is conjugatedto a radioisotope. In a further embodiment, a DBDpp is conjugated to anisotope selected from 90Y, 125I, 131I, 123I, 111In, 105Rh, 153Sm, 67Cu,67Ga, 166Ho, 177Lu, 186Re and 188Re using anyone of a number of knownchelators or direct labeling. In other embodiments, the DBDpp is coupledto drugs, prodrugs or lymphokines such as interferon. Conjugates of theDBDpp and cytotoxin can routinely be made using a variety ofbifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). In a specific embodiment, the toxin isconjugated to a DBDpp fusion protein through an enzyme-cleavable linkersystem (e.g., such as that present in SGN-35). Conjugates of a DBDpp andone or more small molecule toxins, such as a calicheamicin,maytansinoids, a trichothene, and CC1065, and the derivatives of thesetoxins that have toxin activity, can also be used.

In some embodiments, the cytotoxic agent is covalently attached to aDBDpp by a linker. In some embodiments, the linker attaching the DBDppand the cytotoxic agent is cleavable by a protease.

Therapeutic Use as Cell Associated Receptor

In one embodiment of the invention, DBDpp-CAR are used for purposes ofredirecting transduced T cells to a tumor target defined by the bindingspecificity of the DBDpp-CAR. In one example primary T cells aretransduced with a lentiviral vector encoding a CAR that combines a DBDtarget binding domain with a transmembrane domain and an intracellulardomain of CD3-zeta, CD28, 4-1BB. The resultant population of transducedT cells may therefore elicit a DBDpp-CAR-mediated T-cell response. Inone embodiment T cells are genetically modified to express DBDpp-CAR andthe DBDpp-CAR T cell is infused to a recipient in need thereof. Theinfused cell is able to kill tumor cells in the recipient. Severalembodiments of the invention are particularly advantageous because theyinclude one, several or all of the following benefits: (i)target-binding specificity, (ii) enhanced therapeutic efficacy, (iii)reduced off-target side effects, (iv) customizability for markers of aparticular patient or patient population, (v) enhanced stability duringproduction and processing, and (vi) ability to target one, two, or morespecific targets to enhance target-directed therapy.

“Genetically modified cells”, “redirected cells”, “geneticallyengineered cells” or “modified cells” as used herein refer to cells thatexpress a DBDpp provided herein. In a particular embodiment, thegenetically modified cells express a DBDpp fusion protein such as aDBDpp-CAR. In a further embodiment, the genetically modified cellsexpress and display a DBDpp-CAR on the cell surface.

“Disease targeted by genetically modified cells” as used hereinencompasses the targeting of any cell involved in any manner in anydisease by the genetically modified cells, irrespective of whether thegenetically modified cells target diseased cells or healthy cells toeffectuate a therapeutically beneficial result. The genetically modifiedcells include but are not limited to genetically modified T-cells, NKcells, hematopoietic stem cells, pluripotent embryonic stem cells orembryonic stem cells. The genetically modified cells express theDBDpp-CAR, which can target any of the antigens expressed on the surfaceof target cells.

In one embodiment, the DBDpp portion of the DBDpp-CAR is designed totreat a particular cancer. Cancers that can be treated include tumorsthat are not vascularized, or not yet substantially vascularized, aswell as vascularized tumors. The cancers can comprise non-solid tumors(such as hematological tumors, for example, leukemias and lymphomas) orcan comprise solid tumors. Types of cancers to be treated with theDBDpp-CARs include, but are not limited to, carcinoma, blastoma, andsarcoma, and certain leukemia or lymphoid malignancies, benign andmalignant tumors, and malignancies e.g., sarcomas, carcinomas, andmelanomas. Adult tumors/cancers and pediatric tumors/cancers are alsoincluded.

Examples of hematological (or hematogenous) cancers include leukemias,including acute leukemias (such as acute lymphocytic leukemia, acutemyelocytic leukemia, acute myelogenous leukemia and myeloblasts,promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronicleukemias (such as chronic myelocytic (granulocytic) leukemia, chronicmyelogenous leukemia, and chronic lymphocytic leukemia), polycythemiavera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent andhigh grade forms), multiple myeloma, Waldenstrom's macroglobulinemia,heavy chain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma,and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostatecancer, hepatocellular carcinoma, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroidcarcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervicalcancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNStumors (such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pineaioma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

In one embodiment, cancers and disorders can be treated using cellexpressing DBDpp-CAR that target CD19, CD20, CD22, and ROR1. In onespecific embodiment, the DBD-CAR can be designed to target CD22 to treatB-cell lymphoma. In another embodiment the cell expressing DBDpp-CARcontain a DBDpp designed to target CD19 can be used to treat cancers anddisorders including but are not limited to pre-B ALL (pediatricindication), adult ALL, mantle cell lymphoma, diffuse large B-celllymphoma, salvage post allogenic bone marrow transplantation, and thelike.

“B-cell associated diseases” as used herein include B-cellimmunodeficiencies, autoimmune diseases and/or excessive/uncontrolledcell proliferation associated with B-cells (including lymphomas and/orleukemias). Examples of such diseases, wherein DBDpp-CAR may be used fortherapeutic approaches include but are not limited to systemic lupuserythematosus (SLE), diabetes, rheumatoid arthritis (RA), reactivearthritis, multiple sclerosis (MS), pemphigus vulgaris, celiac disease,Crohn's disease, inflammatory bowel disease, ulcerative colitis,autoimmune thyroid disease, X-linked agammaglobulinaemis, pre-B acutelymphoblastic leukemia, systemic lupus erythematosus, common variableimmunodeficiency, chronic lymphocytic leukemia, diseases associated withselective IgA deficiency and/or IgG subclass deficiency, B lineagelymphomas (Hodgkin's lymphoma and/or non-Hodgkin's lymphoma),immunodeficiency with thymoma, transient hypogammaglobulinemia and/orhyper IgM syndrome, as well as virally-mediated B-cell diseases such asEBV mediated lymphoproliferative disease, and chronic infections inwhich B-cells participate in the pathophysiology.

In one embodiment, the DBDpp-CAR can be designed to target mesothelin totreat mesothelioma, pancreatic cancer, ovarian cancer, and the like. Inone embodiment, the DBDpp-CAR can be designed to target CD33/IL3Ra totreat acute myelogenous leukemia and the like. In one embodiment, theDBDpp-CAR can be designed to target c-Met to treat triple negativebreast cancer, non-small cell lung cancer, and the like. In oneembodiment, the DBDpp-CAR can be designed to target PSMA to treatprostate cancer and the like. In one embodiment, the DBDpp-CAR can bedesigned to target Glycolipid F77 to treat prostate cancer and the like.In one embodiment, the DBDpp-CAR can be designed to target EGFRvIII totreat gliobastoma and the like. In one embodiment, the DBDpp-CAR can bedesigned to target GD-2 to treat neuroblastoma, melanoma, and the like.In one embodiment, the DBDpp-CAR can be designed to target NY-ESO-1 totreat myeloma, sarcoma, melanoma, and the like. In one embodiment, theDBDpp-CAR can be designed to target MAGE A3 to treat myeloma, sarcoma,melanoma, and the like. However, the invention should not be construedto be limited to solely to the antigen targets and diseases disclosedherein. Rather, the invention should be construed to include anyantigenic target that is associated with a disease where a DBDpp-CAR canbe used to treat the disease.

In a preferred embodiment, the DBDpp-CAR is expressed in a T cell andprovides a method for treating or preventing cancer, comprising theadministration of host cells expressing DBDpp-CAR to a cancer patient inwhich the cancer cell expresses a tumor antigen on its surface, andwherein the DBDpp specifically binds the target antigen. Exemplarytarget antigens that the DBDpp and DBDpp-CAR bind include, but are notlimited to, CD19, CD123, TSLPR, and CD267.

The DBDpp-CAR-modified T cells can also serve as a type of vaccine forex vivo immunization and/or in vivo therapy in a mammal. Preferably, themammal is a human.

The DBDpp-CAR-modified T cells provided herein can be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as chemotherapeutics,antibodies, cytokines or cell populations. Compositions provided hereinare preferably formulated for intravenous administration that can beadministered one or more times.

“Antigen loss escape variants” as used herein refer to cells whichexhibit reduced or loss of expression of the target antigen, whichantigens are targeted by a CAR provided herein.

Various embodiments of the invention will now be illustrated through thedescription of experiments conducted in accordance therewith. Theexamples that follow are provided to facilitate the practice of thedisclosed embodiments, and are not to be construed as limiting in anyway the remainder of the disclosure. In the examples, reference is madeto the appended figures.

EXAMPLES Example 1. Immunogenicity Assessment of DBDpp

The sequences of DBDpp, particularly those administered to a subjectand/or used in purifying a composition administered to a subject, arepreferably not antigenic with respect to the subject (e.g., human). Insome embodiments, the sequence of the DBDpp does not contain a humanHLA-DR binding motif or cleavage sites for proteasomes andimmune-proteasomes. In particular embodiments, the DBDpp sequence doesnot contain an antigenic sequence as determined by a computer predictionmodel version existent on the filing date of this specification. Inparticular embodiments, the DBDpp sequence does not contain an MHC(class I or class II) binding site sequence as predicted by an algorithmselected from ProPred (see, e.g., Singh, Bioinformatics 17(12):1236-1237(2001)), ProPred1 (Singh, Bioinformatics 19(8):1009-14 (2003)),SYFPEITHI (see, e.g., Schuler, Immunoinf. Meth. in Mol. Biol.409(1):75-93 (2007)), SMM-align (see, e.g., Nielsen, BMC Bioinformatics8:238 (2007)), RANKPEP (see, e.g., Reche, Hum Immunol 63: 701-709.(2004)), or TEPITOPE (see, Sturniolo, Nat Biotechnol 17:555-561 (1999)),wherein the version of the algorithm and the applied database are inexistence on the filing date of this application.

In silico analysis of the amino acid sequence of alpha3D(MGSWAEFKQRLAAIKTRLQALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEALRKEAAAIRDELQAYRH N (SEQ IDNO:49) revealed a 9 amino acid sequence (i.e., LAAIKTRLQ (SEQ IDNO:50)), that shares characteristics with that of high affinity (bindingthreshold less than 6%) and promiscuous (present in greater than 50% ofrelevant alleles) T cell epitopes (Singh, Bioinformatics 17:1236-1237,2001). This epitope resides within an invariant region of some of theDBDpp libraries. Therefore, with the aim of reducing the potential forimmunogenicity, a Q19E substitution was introduced into SEQ ID NO:49.This conserved and surface exposed substitution appeared unlikely tosignificantly disrupt the hydrophobic core (see, e.g., FIG. 1B). Insilico analysis of the resultant sequence (SEQ ID NO:1) yielded lowerimmunogenicity scores.

Example 2. DBDpp Library Design, Construction and Screening

Unlike natural ligands and binding proteins, the synthetic scaffoldsequence of DBD (i.e., SEQ ID NO:1) has no known binding partner. In theconstruction of DBDpp that bind to targets, residues were considered formutation (i.e., randomization within the library) if they wereconsidered to be surface exposed—exhibiting significant solventaccessibility. A variety of methods are available to assess solventaccessibility of defined molecular structures. For example, PyMOL is anopen source software package developed for molecular visualization andanalysis and may be used to calculate solvent accessible surface areausing the method of Lee and Richards (Lee et al., J. Mol. Biol.55:379-400 (1971)). More specifically, using PyMOL (version 1.4.1) withthe “‘dot solvent” set to 1, “solvent radius” to 1.4 Å, and “dotdensity” set to 4, the solvent accessible surface area can be calculatedfor each amino acid of a homology model of the SEQ ID NO:1 based on thetemplate, PDB 2A3D. Table 2 lists the calculated area (in squareangstroms) for each residue, as measured in the context of the domain(area D) and as an isolated amino acid, independent of steric hindrancesimposed by neighboring residues (area I). The relative accessibility ofa residue within the domain (area D) as compared to the isolated state(area I) is represented as a percent value (% A).

TABLE 2 Solvent Accessibility of the Reference Scaffold Sequence (SEQ IDNO:1) position aa Area I Area D % A position aa Area I Area D % A  1 M295.0 156.9 53.2 38 F 316.3 12.6 4.0  2 G 187.1 56.6 30.2 39 E 289.6112.0 38.7  3 S 227.0 23.6 10.4 40 S 227.0 93.7 41.3  4 W 345.8 107.431.1 41 E 285.0 57.9 20.3  5 A 211.9 53.7 25.4 42 L 281.8 21.5 7.6  6 E286.8 104.7 36.5 43 Q 294.3 120.5 41.0  7 F 318.1 7.8 2.5 44 A 209.679.0 37.7  8 K 303.7 142.0 46.8 45 Y 323.7 33.1 10.2  9 Q 293.7 123.041.9 46 K 303.8 70.1 23.1 10 R 341.2 102.9 30.2 47 G 187.2 64.4 34.4 11L 274.1 19.0 6.9 48 K 298.8 149.1 49.9 12 A 211.5 54.8 25.9 49 G 185.66.9 3.7 13 A 211.3 43.4 20.6 50 N 267.4 80.5 30.1 14 I 279.0 18.0 6.5 51P 238.9 92.8 38.8 15 K 300.5 134.3 44.7 52 E 293.1 110.3 37.6 16 T 246.589.7 36.4 53 V 249.2 5.7 2.3 17 R 339.6 131.8 38.8 54 E 290.7 91.0 31.318 L 279.6 27.8 9.9 55 A 211.2 67.2 31.8 19 E 269.3 135.1 50.2 56 L274.5 10.5 3.8 20 A 211.6 61.6 29.1 57 R 336.4 146.8 43.6 21 L 278.0 8.93.2 58 K 306.7 165.1 53.8 22 G 186.9 67.8 36.3 59 E 290.1 114.0 39.3 23G 186.6 47.9 25.7 60 A 211.1 13.2 6.3 24 S 225.9 12.2 5.4 61 A 211.747.6 22.5 25 E 286.9 130.2 45.4 62 A 211.0 56.7 26.9 26 A 210.5 92.443.9 63 I 275.2 26.3 9.5 27 E 281.6 57.7 20.5 64 R 337.5 119.5 35.4 28 L269.8 4.4 1.6 65 D 261.1 106.5 40.8 29 A 211.4 53.0 25.1 66 E 284.7102.5 36.0 30 A 210.7 54.2 25.7 67 L 272.8 7.3 2.7 31 F 317.0 14.8 4.768 Q 277.9 131.3 47.2 32 E 284.7 96.0 33.7 69 A 211.4 40.4 19.1 33 K306.8 158.8 51.8 70 Y 329.3 50.8 15.4 34 E 281.1 83.1 29.5 71 R 341.0149.1 43.7 35 I 276.3 22.9 8.3 72 H 279.1 135.5 48.5 36 A 211.4 58.527.7 73 N 275.1 130.5 47.4 37 A 209.8 51.4 24.5

Domain residues with % A values that are less than 10% to 11% (e.g., F7,L11, I14, L18, L21, S24, L28, F31, I35, F38, L42, Y45, G49, V53, L56,A60, I63, and L67 of SEQ ID NO:1; Table 2A), were considered to berelatively inaccessible to the exterior solvent and were thereforeconsidered interior core residues of the DBD. Conversely, residues with% A values that were greater than 10% to 11% (e.g., G2, S3, W4, A5, E6,K8, Q9, R10, A12, A13, K15, T16, R17, E19, A20, A29, A30, E32, K33, E34,A36, A37, E39, S40, E41, Q43, A44, E52, E54, A55, R57, K58, E59, A61,A62, R64, D65, E66, Q68, A69, and Y70 of SEQ ID NO:1) are predicted tobe located within regions of the polypeptide associated withalpha-helical secondary structure and to occupy positions that havegreater potential for interaction with macromolecular targets ofinterest. These solvent accessible, alpha helical residues wereconsidered to be candidates for the greatest degree of substitutionaldiversity (including conserved and non-conserved substitutions) in thelibrary.

The non-alpha helical residues of the reference scaffold sequencecorrespond to, in several embodiments, positions Ml, L21, G22, G23, S24,E25, A26, E27, Y45, K46, G47, K48, G49, N50, P51, R71, H72, and N73 ofSEQ ID NO:1. In additional embodiments, non-alpha helical residues ofthe reference scaffold sequence correspond to positions Ml, G22, G23,S24, E25, A26, E27, K46, G47, K48, G49, N50, P51, R71, H72, and N73 ofSEQ ID NO:1. These residues were likewise considered to be candidatesfor conserved and non-conserved substitutions in the library.

Some DBDpp libraries were created through the selective or randommutation of specific solvent exposed amino acid sequence positions ofthe DBDpp. In one series of experiments, libraries, referred to hereinas “face” libraries or “F libraries”, were designed such that thesubstituted residues of the reference scaffold structure of thepolypeptide of SEQ ID NO:1 were clustered on a single face of the domainand to create a single, contiguous binding surface. Face libraries wereconstructed for all three faces (F1, F2 & F3) of the structure of thepolypeptide of SEQ ID NO:1. Due to the asymmetry of the domainstructure, each pair of alpha helices—and therefore each face—forms aunique geometric topology (FIGS. 1 and 2). As modeled in the referencescaffold, the large number targeted residues correspond to contiguoussurface area greater than 1400 square angstroms—significantly greaterthan the binding surfaces measured for a survey of non-antibody bindingscaffolds. (Gilbreth et al., Curr. Opin. Struct. Biol. 22:413-420(2012)).

In another of set of experiments, libraries referred to herein as“combined” libraries or “C libraries” were constructed to identify DBDppthat potentially exhibit multi-faceted binding to a target of interest(Table 3 and FIGS. 1 and 2). The combined libraries (C1 and C2) wereconstructed by combining residues from each of the three helices used inthe F series libraries.

In these experiments, a total of 32 residue positions were subjected tomutagenesis. Each mutagenized position is present in at least 2libraries. Furthermore, each mutagenized position is represented in eachof the two library “architectures”; F and C.

TABLE 3 DBDpp Library Sequence Profiles Library Sequence Profile F1MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALRKEAA AIRDELQAYRHN (SEQ ID NO: 2)F2 MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN (SEQ ID NO: 3) F3MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅ X₆₆LQAYRHN (SEQ ID NO: 4) C1MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALGGSEAELAAFX₃₂X₃₃EIX₃₆AFX₃₉X₄₀ELX₄₃AYKGKGNPEVEX₅₅LRX₅₈X₅₉AAX₆₂IRX₆₅X₆₆LQAYRHN (SEQ ID NO: 5) C2MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALGGSEAELAX₃₀FEX₃₃X₃₄IAX₃₇FEX₄₀X₄₁LQX₄₄YKGKGNPEVEALX₅₇X₅₈EAX₆₁AIX₆₄X₆₅ELX₆₈AYRHN (SEQ ID NO: 6) FlLp_(X)MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂NPEVEALRKEAAAIRDE LQAYRHN (SEQ ID NO: 7)F2Lp_(X) MGSWAEFKQRLAAIKTRLEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEALX₅₂X₅₃EAX₅₆AIX₅₉X₆₀EL X₆₃AYRHN (SEQ ID NO: 8)F3Lp_(X) MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAAFEKEIAAFESELQAYZ₂NPEVEX₅₀LRX₅₃X₅₄AAX₅₇IRX₆₀X₆₁ LQAYRHN (SEQ ID NO: 9)C1Lp_(X) MGSWX₅X₆FKX₉X₁₀LAX₁₃IKX₁₆X₁₇LEALZ₁EAELAAFX₃₀X₃₁EIX₃₄AFX₃₇X₃₈ELX₄₁AYZ₂NPEVEX₅₀LFIX₅₃X₅₄AAX₅₇IRX₆₀X₆₁LQAYRHN (SEQ ID NO: 10) C2Lp_(X)MGSWX₅EFX₈X₉RLX₁₂AIX₁₅X₁₆RLX₁₉ALZ₁EAELAX₂₈FEX₃₁X₃₂IAX₃₅FEX₃₈X₃₉LQX₄₂YZ₂X₅₂X₅₃EAX₅₆AIX₅₉X₆₀ELX₆₃AYRHN (SEQ ID NO: 11) X = all amino acid residues Z = amino acidsequence corresponding to loop1 (Z₁) or loop2 (Z₂) as described hereinDBDpp F2NNK Library Construction

An F2 library was constructed that targeted 12 surface-exposed residueson face 2 (helixes 2 and 3) (Table 3 and FIGS. 2C and 2D). This library,designated F2NNK, was created through Kunkel mutagenesis, utilizingoligos containing NNK codons. Libraries were constructed using amodified pComb phagmid vector in which DBDpp are fused at the C-terminusto the N-terminus of M13 pIII. These DBDpp are also fused at theirN-terminus to the C-terminus of the FLAG epitope tag. The entire DBDppfusion protein is under the secretory control of a DsbA signal peptide(FIG. 3B).

DBDpp Trinucleotide Phosphoramidite Library Construction

Subsequent libraries were constructed through Kunkel mutagenesis in thesame modified pComb phagemid vector as the F2NNK library. In severalembodiments, the FLAG tag is optional (see e.g., FIG. 3C), or can bereplaced with another tag. These libraries were constructed usingtrinucleotide phosphoramidite (codon) mixtures. These mixtures weredesigned to exclude termination, cysteine and proline codons andprovided an equal representation of the remaining amino acids. Librarieswere built using all five sequence profiles (F1, F2, F3, C1 & C2) asshown in Table 3.

Selection Using F2_(NNK) Library

The F2NNK DBD library was used in five rounds of selection againstrecombinant, biotinylated, Human 4-1BB/TNFRSF9/CD137-Fc. ELISA screeningof rescued phage revealed that 89 of 95 clones bound 4-1BB/CD137 with anaverage OD 5.3-fold greater than control (IgG Fc). The distribution ofbinding (ELISA absorbance values) for the 89 clones: CD137 0.353(0.134-0.617), control 0.067 (0.056-0.125). Sequencing of individualphage indicated that all 89 clones were identical at the nucleotidelevel. Notably, this clone, named bb10, contained substitutions in only8 (in bold in the sequence below) of the 12 randomized positions ofsequence of SEQ ID NO:1 that are underlined in sequence:

(bb10: SEQ ID NO: 19) MGSWAEFKQRLAAIKTRLEALGGSEAELAAF LG EI W AFEM EL AAYKGKGN PEVEAL GR EAAAIRM ELQAYRHN.Selection Using F1, F2, F3, C1 & C2 Trinucleotide PhosphoramiditeLibraries

Selections were also performed using F1, F2, F3, C1 & C2 trinucleotidephosphoramidite libraries. In most cases, libraries were pooled prior touse in selection. By combining equal volumes of the individuallibraries, Pool F (libraries F1, F2 and F3) and Pool C (libraries C1 andC2) were generated. These libraries were used in selections for DBDppbinders to 4-1BB/CD137 as well as a larger panel of purified recombinant“target”-Fc proteins, including CD47, CTLA4, DR5, KIR, LAG3, OX40, PD1,PD-L1 and TIM3. (Many of these targets are considered immuno-regulatoryfactors (Pardoll et al., Nat. Rev. Cancer 12:252-264 (2012)). Afterincubation of the target with the pools of DBDpp phage libraries, boundphage-target complexes were captured and separated from unbound phagewith protein A beads (target proteins were Fc fusions). After threerounds of selection, rescued phage clones were screened by ELISA forbinding to the selected target.

For each target, approximately 90 DBDpp phage clones were screened byELISA for binding to the target protein and as well as a non-specificcontrol (e.g., IgG1-Fc). Sequencing was performed on individual DBDppclones that exhibited a target-specific binding signal that was 3 foldhigher than the non-specific control. In some instances, for the ELISAscreening plate in which the majority of DBDpp clones were positive, theentire plate was sequenced. Sequence results indicated that, in total,approximately 70% of DBDpp clones were in the correct reading frame andconformed to one of the five anticipated library sequence profiles(Table 3). Sequences that did not conform to an expected profile weretypically composed of either failed sequencing reads, frame-shiftmutations, truncations, concatemerizations or other cloning artifacts.

DBDpp Bind to a Variety of Targets

Table 4 shows the distribution of clones for each of the libraries as afunction of target and binding data. The three sub-tables tally thedistribution for all sequences (top) and those with target-specificbinding ratios equal to or greater than 2 (middle) or 3 (bottom). (Wheresequences are represented by more than one clone, the average bindingvalue is used.) Of the 794 total sequences, 330 are unique clones, ofwhich 278 yielded an ELISA signal 3 fold above background.

TABLE 4 Distribution of DBDpp clones and unique sequences (T-Total;U-Unique) CD137 CD47 CTLA DR5 KIR LAG3 OX40 PD1 PDL1 TIM3 Library T U TU T U T U T U T U T U T U T U T U T U ALL Sequences Fl 182  78  1  1  1 1 74 26  5  3  3  3  51 30 47 14 F2  7  7  5  5 2 2 F3 416 223 114 49 55 34 74 2  2  1 17 16  3  3  53 41 76 63 22 14 Cl  4  4 1  1  1  1 2 2C2  95  17  3  2  73 12 14  2  5  1 F2^(NNK)  90  1  90  1 Total 794 330208 53 130 48 74 2 90 29 28 21 11 11 4 4 104 71 76 63 69 28 Sequenceswith ELISA ratios greater or equal to 2 Fl 162  64  1  1  1  1 74 26  5 3  2  2 43 24 36  7 F2  7  7  5  5 2 2 F3 402 409 114 49  52 31 74 2  2 1 15 14  3  3 52 40 75 62 15  7 Cl  3  3  1  1 2 2 C2  92  14  3  2  70 9 14  2  5  1 F2^(NNK)  90  1  90  1 Total 756 298 208 53 123 41 74 290 29 26 19 10 10 4 4 95 64 75 62 51 14 Sequences with ELISA ratiosgreater or equal to 3 Fl 159  61  1  1 74 26  5  3 2 2 42 23 35  6 F2  5 5 4 4 1 1 F3 310 195 114 49  51 30  2  1 14 13 3 3 49 38 66 55 11  6 Cl 2  2  1  1 1 1 C2  92  14  3  2  70  9 14  2 5 1 F2^(NNK)  90  1  90  1Total 658 278 207 52 122 40 90 29 25 18 9 9 2 2 91 61 66 55 46 12

Table 5 lists exemplary sequences derived from the experiments describedabove. For each sequence, the Target, library of origin (Lib.), numberof screening occurrences (Count) and target to background ratio (ELISAratio) are indicated. In several embodiments, DBDpp with at least 80%,at least 85%, at least 90%, at least 92%, at least 95% or at least 98%homology to those described above (and elsewhere herein) retainsignificant functional equivalence. In several embodiments, this isadvantageous as the divergence in homology may present certainadvantages, such as reduced immunogenicity, increased cross-reactivity,increased specificity, etc.

TABLE 5Sequences of DBDpp Library Clones Isolated from the Screened LibrariesSEQ ELISA ID NO: Sequence Target Lib. Count ratio 12MGSWVEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFES CD137 F3 3 20.606ELQAYKGKGNPEVEKLRQRAAFIRFRLQAYRHN 13MGSWVEFANRLWAIDQRLFALGGSEAELAAFEKEIAAFESE CD137 F3 7 16.055LQAYKGKGNPEVEHLRDQAAFIRHKLQAYRHN 14MGSWYEFRHRLWAIDQRLYALGGSEAELAAFEKEIAAFES CD137 F3 4 12.974ELQAYKGKGNPEVEGLREAAAFIRAKLQAYRHN 15MGSWYEFSMRLWAIDQRLYALGGSEAELAAFEKEIAAFES CD137 F3 2 12.040ELQAYKGKGNPEVEALRAKAAYIRWKLQAYRHN 16MGSWFEFNHRLWAINERLYALGGSEAELAAFEKEIAAFESE CD137 F3 4 11.925LQAYKGKGNPEVERLRSMAAFIRYKLQAYRHN 17MGSWYEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFES CD137 F3 3 7.707ELQAYKGKGNPEVEYLRETAAHIRTRLQAYRHN 18MGSWYEFHYRLHAIDQRLYALGGSEAELAAFEKEIAAFESE CD137 F3 3 7.262LQAYKGKGNPEVEELRIKAAFIRDRLQAYRHN 19MGSWAEFKQRLAAIKTRLEALGGSEAELAAFLGEIWAFEME CD137 F2 90 5.269LAAYKGKGNPEVEALGREAAAIRMELQAYRHN 20MGSWYEFDLRLHAIYDRLVALGGSEAELAAFEKEIAAFESE CD47 F3 2 14.087LQAYKGKGNPEVEILRDNAAYIRQMLQAYRHN 21MGSWTEFTYRLSAIEWRLWALGGSEAELAWFEQKIAFFED CD47 C2 24 12.517FLQYYKGKGNPEVEALKHEAGAILNELMAYRHN 22MGSWAEFDHRLHAIRERLHALGGSEAELAAFEKEIAAFESE CD47 F3 3 11.651LQAYKGKGNPEVEILRGNAAYIRALLQAYRHN 23MGSWTEFVGRLAAIEFRLWALGGSEAELAWFEAHIAFFED CD47 C2 3 8.230YLQWYKGKGNPEVEALREEAGAIMEELKAYRHN 24MGSWTEFYSRLEAIWVRLQALGGSEAELAMFEDRIAHFEW CD47 C2 37 4.578FLQQYKGKGNPEVEALHEEAIAIRKELAAYRHN 25MGSWHEFHDRLQAIHERLYALGGSEAELAAFEKEIAAFESE CTLA4 F3 73 2.950LQAYKGKGNPEVESLRIAAAHIRQVLQAYRHN 26MGSWNYFKDHLAWIKNSLEALGGSEAELAHFETAIASFER DR5 F1 12 12.993QLQEYKGKGNPEVEALRKEAAAIRDELQAYRHN 27MGSWLYFKEHLAHIKAWLEALGGSEAELAHFELAIADFEYH DR5 F1 5 12.309LQEYKGKGNPEVEALRKEAAAIRDELQAYRHN 28MGSWVYFKEHLAWIKTELEALGGSEAELAHFEHSIADFEMS DR5 F1 4 12.117LQFYKGKGNPEVEALRKEAAAIRDELQAYRHN 29MGSWFYFKQHLAWIKSYLEALGGSEAELAHFERAIAAFEQ DR5 F1 5 11.836HLQMYKGKGNPEVEALRKEAAAIRDELQAYRHN 30MGSWHYFKDHLAEIKGLLEALGGSEAELAHFEMAIADFEHN DR5 F1 5 11.436LQYYKGKGNPEVEALRKEAAAIRDELQAYRHN 31MGSWHYFKGHLAEIKNHLEALGGSEAELAHFERAIAAFERS DR5 F1 7 10.822LQWYKGKGNPEVEALRKEAAAIRDELQAYRHN 32MGSWIYFKEHLAYIKKELEALGGSEAELAHFESAIAVFESTL DR5 F1 4 10.677QYYKGKGNPEVEALRKEAAAIRDELQAYRHN 33MGSWTYFKEHLAEIKYMLEALGGSEAELAHFEVAIADFEKM DR5 F1 8 10.256LQYYKGKGNPEVEALRKEAAAIRDELQAYRHN 34MGSWWLFKDHLAEIKTALEALGGSEAELAHFEMAIAAFEKQ DR5 F1 3 9.748LQYYKGKGNPEVEALRKEAAAIRDELQAYRHN 35MGSWSEFYNRLDAIESRLLALGGSEAELALFEIQIARFEKVL KIR C2 5 8.399QAYKGKGNPEVEALRGEARAIFAELYAYRHN 36MGSWYEFYNRLYAIEIRLYALGGSEAELAAFEKEIAAFESEL KIR F3 2 4.244QAYKGKGNPEVERLRVRAAKIRVILQAYRHN 37MGSWLWFKIFLAEIKYFLEALGGSEAELAAFDFEIHAFHVEL KIR C1 1 4.170FAYKGKGNPEVEVLREVAAEIRWDLQAYRHN 38MGSWTEFQSRLDAIHSRLRALGGSEAELAAFEKEIAAFESE PD-L1 F3 2 8.682LQAYKGKGNPEVELLRDDAAFIRHFLQAYRHN 39MGSWQEFDDRLNAIKARLQALGGSEAELAAFEKEIAAFESE PD-L1 F3 2 7.413LQAYKGKGNPEVEDLRDDAAFIRRFLQAYRHN 40MGSWYEFQNRLHAIHERLNALGGSEAELAAFEKEIAAFESE PD-L1 F3 2 6.345LQAYKGKGNPEVELLRDDAAFIRHFLQAYRHN 41MGSWFEFQDRLTAINERLSALGGSEAELAAFEKEIAAFESE PD-L1 F3 2 6.015LQAYKGKGNPEVETLRSDAAFIRRFLQAYRHN 42MGSWYEFESRLDAIHERLHALGGSEAELAAFEKEIAAFESE PD-L1 F3 6 4.882LQAYKGKGNPEVENLRGDAAFIRHFLQAYRHN 43MGSWYEFNHRLDAISKRLNALGGSEAELAAFEKEIAAFESE PD-L1 F3 2 2.982LQAYKGKGNPEVEELRGDAAFIRHFLQAYRHN 44MGSWFEFENRLHAIVHRLGALGGSEAELAAFEKEIAAFESE PD-L1 F3 2 2.764LQAYKGKGNPEVETLRADAAFIRHYLQAYRHN 45MGSWVVFKVDLATIKYILEALGGSEAELAEFEGEIAGFEYSL TIM3 F1 2 5.788QFYKGKGNPEVEALRKEAAAIRDELQAYRHN 46MGSWTIFKEWLAFIKTDLEALGGSEAELAFFEGWIASFEME PD1 F1 14 17.145LQKYKGKGNPEVEALRKEAAAIRDELQAYRHN 47MGSWVMFKVVLLADIKSHLEALGGSEAELAFFEGFIAAFETH PD1 F1 4 8.132LQVYKGKGNPEVEALRKEAAAIRDELQAYRHN 48MGSWYAFKDYLADIKGWLEALGGSEAELAFFEIFIARFELEL PD1 F1 2 3.295QAYKGKGNPEVEALRKEAAAIRDELQAYRHN

N-terminal FLAG tag fusions of pb04 (SEQ ID NO: 182) and a3D (SEQ ID NO:49) were expressed and purified from E. coli cultures. Throughassessment by ELISA, purified FLAG-pb04 binds in a dose dependent mannerto PD-L1-Fc coated microtiter wells. In contrast, FLAG-a3D exhibits nodetectable binding to the PD-L1-Fc target protein (FIG. 3D). The resultsdemonstrate that modification of reference scaffold sequence (SEQ IDNO:1) is effective in providing a robust source of DBDpp that are ableto bind, with novel specificity, a diverse set of targets of interest.

Example 3. DBDpp Fusion Proteins

To assess the modular nature of DBDpp as a binding element, the DBDppCD137-binder, bb10 (SEQ ID NO:19), was reformatted as a fusion to eitherthe N or C terminus of the heavy chain of an antibody derived from thesequence of the RSV-specific monoclonal antibody palivizumab (SYNAGIS®)(shown schematically in FIGS. 4A and 4B, respectively). As a comparatoranalogous fusions were generated using the DBDpp parental sequence (SEQID NO:1), which is not known to exhibit any binding specificity.Proteins were produced in HEK293F suspension cells that were transientlytransfected with equimolar ratios of independent heavy chain-bb10 fusionand light chain cDNA expression constructs, and purified throughconventional protein A affinity methods. Separation by SDS-PAGE ofpurified samples indicated that the migration of heavy chain-bb10(DBDpp) fusion proteins were commensurate with predictions based onmolecular weight (data not shown).

Analysis by SEC indicated that the bb10 DBDpp antibody fusions were notaggregated and migrated as predicted relative to the size standards(data not shown). SYN-bb10 and bb10-SYN fusions demonstrate similarmigration to each other, both ran faster than the parental SYNAGIS®antibody.

Bi-specific antibodies, SYN-bb10 and bb10-SYN exhibit binding to bothCD137 and RSV (FIG. 4C-D; closed squares are bb10-SYN, closed circlesare SYN-bb10)), demonstrating that a novel binding activity was impartedto the parental DBD sequence and the functionality of DBDpp is retainedas both N and C-terminal fusion. In contrast, fusions between atargetless alpha-helical protein scaffold and SYN (DBD-SYN forN-terminal fusion, open circles; SYN-DBD for C-terminal fusion, opensquares) showed binding only to RSV, but no binding to CD137 wasimparted.

Binding of DBDpp, bb10 to CD137 is demonstrated using two differentexperimental methods: ELISA (FIG. 4C-D) and FACS (FIG. 6A-C). In theseassays the target antigen is presented and ultimately recognized inthree different formats: either directly bound to plastic (FIG. 4C-D) orin situ, as part of a cell membrane (FIG. 6A-C).

Treatment of PBMCs with SYN-bb10 and bb10-SYN demonstrates that inaddition to binding, both fusion proteins are capable of inducing adownstream biological response in target cells (FIG. 7A-D).

In vivo stability is critical to the clinical efficacy of mostbiotherapeutics. Pharmacokinetic measurements of bb10 fusions (SYN-bb10and bb10-SYN) were performed to assess the relative stability of DBDppas compared to the mAb fusion partner (FIG. 8). The in vivo stabilitywas determined by analysis of both the RSV and CD137 binding of thebi-specific antibodies present in serum from CD1 mice that received asingle intravenous injection (1 mg/kg) of the fusion proteins. Serumsamples were collected at 15 minutes and 48 hours, and were assayed byELISA. Both N-terminal and C-terminal DBDpp fusion proteins demonstratesustained stability in vivo.

Example 4. Use of DBDpp in Affinity Purification

Eight CD137 binding DBDpp ligands (SEQ ID NO:12-19) were reformatted asN-terminal hexahistidine fusion proteins. Their tagged sequences andcorresponding parent sequences are shown in Table 5.

TABLE 5 N-terminal hexahistidine fusion proteins prepared from SEQ ID NO: 12-19. Parent New SEQ  Seq ID NO: IDHis-Fusion Protein Sequence 12 51 MGSWVEFGHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEKLRQRAAFIR FRLQAYRHNGGGGSHHHHHH 13 52MGSWVEFANRLWAIDQRLFALGGSEAELAAFE KEIAAFESELQAYKGKGNPEVEHLRDQAAFIRHKLQAYRHNGGGGSHHHHHH 14 53 MGSWYEFRHRLWAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEGLREAAAFIR AKLQAYRHNGGGGSHHHHHH 15 54MGSWYEFSMRLWAIDQRLYALGGSEAELAAFE KEIAAFESELQAYKGKGNPEVEALRAKAAYIRWKLQAYRHNGGGGSHHHHHH 16 55 MGSWFEFNHRLWAINERLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVERLRSMAAFIR YKLQAYRHNGGGGSHHHHHH 17 56MGSWYEFGHRLWAIDQRLYALGGSEAELAAFE KEIAAFESELQAYKGKGNPEVEYLRETAAHIRTRLQAYRHNGGGGSHHHHHH 18 57 MGSWYEFHYRLHAIDQRLYALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEELRIKAAFIR DRLQAYRHNGGGGSHHHHHH 19 58MGSWAEFKQRLAAIKTRLEALGGSEAELAAFL GEIWAFEMELAAYKGKGNPEVEALGREAAAIRMELQAYRHNGGGGSHHHHHH

Each fusion was expressed separately in E.Coli BL21 (DE3) cells. Aftercell lysis and purification using immobilized metal ion chromatographyeach of the CD137 binding DBDpp ligands were re-purified usingreverse-phase HPLC. The HPLC columns (20×100 mm C-4, Western Analytical)were each eluted with 0.1% TFA:Acetonitrile (88:12 for 2 minutesfollowed by a linear gradient to 58:42 at 15 minutes). The major peak atapproximately 12 minutes corresponded to the target ligand (see arrow inFIG. 9). The purified ligands were lyophilized prior to further use.

The identity and purity of each of the ligands was confirmed byelectrospray mass spectrometry (Table 6) and SDS-PAGE (FIG. 10). Table 6shows the calculated molecular weight for each of the CD137 targetingDBDpp, based on their sequence. Table 6 also shows the expectedmolecular weight for each of the DBDpp after replacement of theN-terminal methionine with a hexahistidine tag. Table 6 also shows theobserved molecular weights. For each of the CD137 targeting DBDpps, withthe exception of SEQ ID NO. 54, the observed molecular weight correlatedwell with the expected molecular weight, indicating that the dominantspecies in the purified sample included the hexahistidine tag. For SEQID NO: 54 the species containing the methionine was the major species.

TABLE 6 Comparison of expected molecular weight and observed molecularweight for the 8 proteins SEQ ID 51-58. Expected MWt Seq ID Calc. MWt(without Met) Observed MWt 51 9596 9465 9464 52 9501 9370 9369 53 95009369 9368 54 9569 9438 9568 55 9651 9520 9518 56 9585 9454 9453 57 96439512 9511 58 9207 9076 9075

FIG. 10 shows a Coomassie blue stained SDS-PAGE analysis of each of theCD137 targeting DBDpps with a hexahistidine tag. Lane 1 is a molecularweight ladder (kilodaltons), lane 2 is SEQ ID NO: 58, and lanes 3-9correspond to SEQ ID NOS: 51-57, respectively. Each of the lanes shows aclean and precise band without smearing or evidence of smaller bandsthat would suggest breakdown of the DBDpp.

FIG. 11 shows the deconvoluted electrospray mass spectrum of the DBDppaccording to SEQ ID NO: 54, in which it was clarified that the dominantspecies in the sample remained protein with the N-terminal methionine,as opposed to the His6 tag.

In accordance with several embodiments disclosed herein, the productionmethods disclosed herein for production of tagged DBDpp result in thedominant species (e.g., greater than 50%, greater than 60%, greater than70%, greater than 80%, etc.) of a given production run being the taggedspecies. As discussed above, tags other than His6 may be used, dependingon the embodiment.

The target protein, CD137, was constructed as a CD137-Fc-His6 fusionprotein. A (Leu24-Gln186)/rHuman Fc (Lys100-Lys329) chimera (SEQ ID: 59)with hexahistidine tag was prepared by transient transfection of HEK293cells and purified from the clarified cell supernatant using immobilizedmetal ion chromatography (IMAC) and then buffer exchanged into PBS. Thismaterial behaved identically to authentic protein purchased fromcommercial sources (RnD systems) and was used as a positive control forCD137.

SEQ ID: 59 is shown below:

LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQDIEGRMDKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK HHHHHH

The binding of the eight his-tagged DBDpp ligands (SEQ ID NO:51-58) tothe CD137 target protein was assessed using biolayer interferometry(ForteBio, Menlo Park, Calif.) according to established methods. Thebinding assay was constructed by immobilizing the CD137-Fc-His6 viaprotein A and then incubating in solutions of each of the DBDpp ligandsfor 5 minutes to measure the association (binding) phase. The sensorswere then placed in buffer to monitor the dissociation phase and thecomplete sensorgrams (time in seconds on the X-axis and nm on theY-axis) are shown in FIGS. 12A, 12C, 12E, 12G, 12I, 12K, 12M, and 12Ofor DBDpps of SEQ ID Nos: 51-58, respectively. The data was fitted bysteady state analysis, which is depicted in each of FIGS. 12D, 12D, 12F,12H, 12J, 12L, 12N, and 12P for the corresponding DBDpp. The datarevealed yield affinity constants for CD137 in the range 140 nM-1.7 μM(Table 7).

TABLE 7 Calculated affinity constants for the 8 ligands SEQ ID 51-59.SEQ ID K_(D) (M) 51 6.4 x 10⁻⁷ ± 1.8 x 10⁻⁷ 52 4.6 x 10⁻⁷ ± 8.5 x 10⁻⁸53 1.4 x 10⁻⁷ ± 3.0 x 10⁻⁸ 54 4.0 x 10⁻⁷ ± 9.4 x 10⁻⁸ 55 1.7 x 10⁻⁷ ±4.0 x 10⁻⁸ 56 7.7 x 10⁻⁷ ± 1.2 x 10⁻⁷ 57 1.0 x 10⁻⁶ ± 1.7 x 10⁻⁷ 58 1.7x 10⁻⁶ ± 2.6 x 10⁻⁷

Four of the eight his-fusion proteins were coupled to NHS-activatedSepharose 4 Fast Flow (GE Healthcare Life Sciences) using themanufacturer's guidelines for 4 hours at room temperature and thenwashed before assaying the ligand density (Table 8).

TABLE 8 Measured ligand density of affinity resins Ligand coupling SeqID concentration mg/ml Resin Ligand Density mg/ml 51 4.9 10 52 1.7 3.353 3.9 7 56 3.3 6.5 58 3.9 5.4

For each of the resins a portion of the washed resin was packed into a3×25 mm glass column (Omnifit) and fitted to a BioLogic chromatograph(Bio-Rad). The resins were equilibrated with 1 ml of phosphate bufferedsaline (PBS) at a flow rate of 0.5 mL/min. A sample of Chinese hamsterovary (CHO) cell supernatant containing CD137 (which had been added to aconcentration of 0.25 mg/ml) was applied to the column (550 ug ofCD137-Fc-His at 0.2 mL/min) and followed by washing (7.5 ml with PBS at0.5 ml/min) and elution (2 ml with 50 mM Na Acetate pH 3 at 0.2 ml/min).Prior to evaluating binding of another sample, resins were regeneratedwith 1 ml with 6M Guanidine-HCl at 0.5 ml/min and re-equilibrated with25 column volumes with PBS at 0.5 ml/min.

FIG. 13 depicts the chromatogram form the purification of CD137-Fc-His6protein from the CHO supernatant. The peak eluting at 8-9 mL correspondsto the eluted CD137-Fc-His6 protein. This data demonstrates that theDBDpp disclosed herein can successfully be used to capture a targetprotein from a sample with a high degree of specificity, even when theinput sample comprises a complex mixture of biological proteins thathave the potential to interfere with target-binder interactions.

To further assess the specificity of binding, the fractions from theelution phase were assessed by SDS-PAGE to confirm that the affinityresin was able to purify CD137 while allowing removal of a considerableproportion of the host cell protein. FIG. 14A shows a Coomassie bluestained gel with a molecular weight ladder in Lane 1 (kilodaltons). Lane2 shows a positive control IMAC-purified CD137 protein. Lane 3 depicts anegative control which is CD137-Fc-His6 spiked into CHO supernatant at a0.25 mg/mL concentration, which represents lack of purification of theCD137 from the supernatant. Lane 4 is eluate after purification with theDBDpp of SEQ ID NO. 58, lane 5 is eluate after purification with theDBDpp of SEQ ID NO. 51, lane 6 is eluate after purification with theDBDpp of SEQ ID NO. 52, lane 7 is eluate after purification with theDBDpp of SEQ ID NO. 57, and lane 8 is eluate after purification with theDBDpp of SEQ ID NO. 57. These data show that the DBDpp specificallypurify target proteins, of which CD137 is a non-limiting example. Lane 3clearly shows additional protein matter in the lane, which is a resultof the protein components of the CHO cell supernatant. In contrast lanes4-8 show clean bands, with little or no other protein species, which issimilar to the positive control IMAC purified CD137.

Additionally, the Western blot analysis shown in FIG. 14B furtherreinforce the ability of the DBDpp described herein to isolate aspecific target protein from a complex protein-containing sample. Thelayout of the lanes is the same as that as described above for FIG. 14A.An anti-penta-His-HRP conjugate antibody (Qiagen) was used fordetection. As initially suggested by the Coomassie stain, the Westernblot analysis confirms that the DBDpp described herein can successfullybind a target protein with specificity, and then be used for specificisolation of that protein from a complex starting sample. Each of theCD137 targeting DBDpp in Lanes 4-8 show the ability to purify CD137 aswell, if not better, as purification with immobilized metal ionchromatography (compare lanes 4-8 with lane 2). As CD137 is just one,nonlimiting embodiment, the DBDpp disclosed herein, according toadditional embodiments can be used to purify a wide variety of targetproteins from various starting samples. In some embodiments, thestarting sample is a biological fluid while in other embodiments, othertypes of liquid or gaseous samples make up the starting material fromwhich a target protein is to be captured and purified.

Example 5. Characterization of DBDpp Stability

The present example was performed to evaluate the stability of DBDppaccording to embodiments disclosed herein.

DBDpp-6xHIS fusions (pb04 and pb06) and scFv-6xHIS fusions wereexpressed through in vitro transcription and translation reactions (NEB,PureExpress). Samples were diluted in ELISA blocking buffer (ThermoFisher) 30 fold. Individual samples were subsequently incubated ateither 25° C., 40° C., 55° C., 70° C. or 100° C. for 2 minutes and thenrapidly returned to room temperature. The temperature increase, followedby rapid cooling can cause denaturation of the protein or otherbreakdown of the three-dimensional structure of proteins that reducetarget interaction and/or binding. The ability of proteins to bind witha ligand, such as a target tumor antigen, after being exposed todenaturing conditions indicates either enhanced stability duringexposure to elevated temperature or an ability to re-fold and maintainfunction after exposure. Regardless, the elevated thermal stabilitymakes such proteins attractive candidates for maintaining functionduring and after the rigors of production, storage, thawing and clinicaluse.

The heat-exposed samples were serially diluted in ELISA buffer andmeasured for binding to PD-L1-Fc in ELISA. Bound proteins were detectedwith HRP-conjugated rabbit anti-6xHIS polyclonal antibody (Abcam). FIG.15A depicts the binding of DR5 scFv to cells expressing PD-L1. Sincethis particular scFv is not configured to bind PD-L1, this function as anegative control, and as expected, no binding is detected for any of theDR5 scFv, regardless of the temperatures they were exposed to. FIG. 15Bshows that a scFv directed against PD-L1 will successfully bind to itstarget after exposure to elevated temperatures, up to about 55° C.However, exposure to temperatures of 70° C. show a decrease in theability of the scFv to successfully bind to PD-L1, indicative of theheat labile nature of scFv. After heating the scFv to 100° C., bindingof target PD-L1 is completely eliminated.

In contrast, DBDpp exhibit improved thermal stability. FIG. 15C depictsdata exhibiting the ability of a DBDpp (pb04 in this nonlimitingembodiment) to bind to target PD-L1, even after being exposed toelevated temperatures of 100° C. FIG. 15D shows similar data for adifferent, nonlimiting embodiment of a DBDpp (pb06). Again, the DBDppretain the ability to bind its target after exposure of temperatures upto 100° C.

These data suggest that DBDpp, according to several embodimentsdisclosed herein, have the ability to resist denaturation and/or refoldafter exposure to elevated temperatures, and still retain the ability tobind a desired target. As discussed above, this makes DBDpp attractivetargeting moiety is as their increased thermal stability suggests theyare robust enough to handle manufacturing processes (or otherproduction/handling protocols) which may involve elevated temperaturesbetter than other types of targeting moieties, such as scFv.

Example 6. DBDpp Species Cross Reactivity

The present example was performed to establish the species specificityof DBDpp as disclosed herein.

FIG. 16A establishes that a soluble DBDpp selected for binding to humanPD-L1 (pb04 in this nonlimiting embodiment), can also bind PD-L1 ofcynomolgus (Macaca fascicularis), as assessed by ELISA. Human PD-L1 orcynomolgus PD-L1 was immobilized in separate wells of an ELISA plate.Soluble DBDpp (FLAG tagged) directed against PD-L1 was incubated in therespective plates to assess the degree of binding with PD-L1 from eachspecies. As shown, pb04 DBDpp binds to both human and cynomolgus PD-L1.FIG. 16B further demonstrates that PD-L1 of human and cynomolgus originis bound by a CAR comprising a DBDpp (pb04 in this nonlimitingembodiment) expressed on the surface of human T cells. As exhibited bythe flow cytometry data PD-L1-directed DBDpp CAR T cells can bindsoluble human PD-L1 and cynomolgus PD-L1 (97.7% for human, and 97.1% forcynomolgus).

Example 7. CAR T Cells Expressing DBDpps Bind Target Molecules

The present example was performed to establish the ability oftransiently expressed CARs comprising DBDpp in this nonlimitingembodiment, to bind a tumor target comprising an amino acid sequencethat is at least 95% identical to residues 19-305 of SEQ ID NO: 187(CD123).

293T cells were transiently transfected with pcDNA3 expression vectorsencoding DBDpp containing CARs using (see, e.g., FIG. 5B) Lipofectamine3000 (Life Technologies). After 24 hours, cells were collected usingCellStripper™. Cells were assessed for CAR expression using afluorescent-labeled anti-FLAG antibody. DBDpp containing CARs binding toCD123 was measured by incubating cells with a Fc-fusion proteincomprising the extracellular domain of CD123 fused to human IgG1 Fc incell culture media at 37° for 30 minutes, washing, and then detectionwith a PE anti-human IgG antibody. Both CD123 binding and CAR expression(FLAG Tag) were assayed by flow cytometry. Data are shown in FIG. 17,and each data point indicates the average FLAG expression andCD123-binding for each of the DBDpp-CARs from multiple experiments.

FIG. 17 summarizes flow cytometry data demonstrating that CARscomprising a CD123 binding scFv (32716) or a DBDpp expressed in human Tcells bind soluble CD123, as a non-limiting embodiment of a target ofinterest. The X-axis is a measure of CAR expression on human T cells.The Y-axis represents CAR binding of the Fc-CD123.

Example 8. T Cells Expressing CARs Comprising DBDpp (DBDpp-CAR) InduceIntracellular Signaling

To assess the ability of CARs comprising a DBDpp (DBDpp-CAR) to initiatesignal transduction, a Jurkat reporter cell line, containing NuclearFactor of Activated T-cells (NFAT) enhancer coupled to luciferasereporter gene, was stably expressed in Jurkat cells. Various CARconstructs were electroporated into the Jurkat reporter cell line. After24 hours post-electroporation, CAR expression was assessed by detectionwith a fluorescent labeled anti-FLAG monoclonal antibody. TheDBDpp-CAR-expressing Jurkat cells were then co-cultured with CD123+(BDCM, acute myelogenous leukemia, as a non-limiting embodiment) tumorcells for 6 hours after which NFAT mediated signaling was measuredthrough the addition to the cells of luciferase assay reagent (Promega)and quantitation of relative luminescence units (RLU), as shown in FIG.18.

These data demonstrate that, in this non-limiting embodiment, DBDpp-CARsare expressed on cells (e.g., human T cells), and when so expressed, caninitiate an intracellular signaling cascade following exposure to atarget (Fc-CD123 in this non-limiting embodiment) bound by the DBDpp.

Example 9. DBDpp-CARs Expressed in Human T Cells Produce Cytokines onTarget Binding

Engagement of a target by a CAR-expressing T cell can result in cytokinesecretion.

Accordingly, 293T cells were transiently transfected with 3rd generationlentiviral packaging vectors (pRSV-REV, pMDLg/pRRE, and pMD2.G) withpELNS vectors encoding DBDpp-CARs using Lipofectamine 3000. Six hourspost-transfection the media was changed, then lentivirus containingmedia was collected at 30 and 54 hours post-transfection, pooled, thencentrifuged to remove cell debris. Lentivirus was then aliquoted andstored at −80° C. until used for viral transduction. Transduction ofhuman T-cells with CAR lentivirus was performed using total human PBMCs,activated with αCD3/CD28 T-cell activation beads in culture mediasupplemented with 40 U/ml of IL-2. After 24 hours, 2×10⁶ PBMCs wereplated per well in a 6-well tissue culture plate with 1 ml of culturemedia and 3 ml of lentivirus containing media supplemented with 40 U/mlof IL-2 and protamine sulfate. Plates were then centrifuged for 2 hoursat 1000×g at 32° C. and then incubated overnight 37° C. The followingday the lentivirus transduction procedure was repeated with freshculture media and lentivirus-containing media. 72 hours after theinitial cell activation, T-cell activation beads were removed, thenT-cells were cultured for expansion at −0.25-0.5×10⁶ T-cells/ml in freshmedia supplemented with 100 U/ml of IL-2. Every 2-3 days T-cells weresupplemented with additional T-cell media and IL-2, until they were usedfor the cytokine assays (described below) 7-10 days after the initialactivation.

Cytokine production in response to target antigen expression (CD123 inthis non-limiting embodiment) was assessed by culturing 25,000transduced T-cells (7 days post-activation) with 25,000 non-target(K562, CD123−) or target (BDCM, CD123+) tumor cells per well in 96-wellplates. After 24 hours culture supernatants were collected and cytokineproduction was assessed by ELISA. Culture supernatants were diluted 1:5prior to ELISA. Similarly, cytokine production in response to PD-L1target antigen expression was assessed by culturing 25,000 transducedT-cells (7-days post activation) with 25,000 non-target (K562, PD-L1−)or target (SUDHL-1, PD-L1+) tumor cells per well in 96-well plates.After 24 hours culture supernatants were collected and cytokineproduction was assessed by ELISA. Culture supernatants were diluted 1:5prior to ELISA.

FIG. 19A demonstrates that T cells expressing CD123 binding DBDpp-CARsproduce interferon gamma (IFNγ) following stimulation with CD123+ BDCMcells, but not the CD123-cell line K562. FIG. 19B demonstrates that Tcells expressing CD123 binding DBDpp-CARs produce interleukin 2 (IL2)following stimulation with CD123+ BDCM cells, but not the CD123-cellline K562. FIG. 20A demonstrates that T cells expressing PD-L1 bindingDBDpp-CARs produce interferon gamma (IFNγ) following stimulation withPD-L1+ SUDHL-1 cells but not the PD-L1− cell line K562. FIG. 20Bdemonstrates that T cells expressing PD-L1 binding DBDpp-CARs produceinterleukin 2 (IL2) following stimulation with PD-L1+ SUDHL-1 cells butnot the PD-L1− cell line K562.

Example 10. T Cells Expressing DBDpp-CARs Proliferate when Co-Culturedwith Target-Expressing Tumor Cells

T cells expressing target-binding CARs can proliferate followingengagement of soluble target or target expressing tumor cells.

Proliferation of DBDpp-CAR transduced human T cells in response to tumorcells expressing target antigen (of which CD123 is a non-limitingexample) was assessed by culturing transduced T-cells (1×10⁵, day 10post-activation) with 1×105 mitomycin-C pre-treated tumor cells in24-well plates. Tumor cells included non-target expressing K562(CD123-), intermediate target-expressing lines KG1a and MOLM-13(CD123-intermediate), and BDCM (CD123-high). Transduced T cells werecollected and counted after 96 hours of co-culturing with tumor cells.This approach was also used in assessing T-cell proliferation inresponse to tumor cells expressing PD-L1 target antigen. Tumor cellsincluded non-target expressing K562 (PD-L1−), intermediatetarget-expressing lines BDCM and H460 (PD-L1-intermediate), and SUDHL-1(PD-L1-high). Cells were collected and counted after culturing for96-hours. Data from these proliferation experiments is shown in FIG. 21and FIG. 22 respectively.

The bars of the histogram in FIG. 21 represent (moving from left toright): culture of DBDpp-CAR T cells alone, co-culture with CD123negative K562 cells, co-culture with low level CD123 expressing KG1acells, co-culture with low level CD123 expressing MOLM-13 cells, andco-culture with high-level CD123 expressing BDCM cells. As indicated bythe height of the histogram bars, when incubated with cells that expressCD123 (in intermediate or high levels), there is a correspondingincrease in T cell proliferation. These data indicate DBDpp-CAR T cellstargeting CD123, proliferate in response to binding CD123 comparable to,or in some embodiments to a greater degree than, CD123 targeting scFv.Similarly, in FIG. 22, the bars of the histogram represent (moving fromleft to right): culture of PD-L1-DBDpp-CAR T cells alone, co-culturewith PD-L1 negative K562 cells, co-culture with intermediate level PD-L1expressing BDCM cells, co-culture with high level PDL2 expressingSUDHL-1 cells, and co-culture with intermediate level PD-L1 expressingH460 cells. These data show a specificity of the response of the T cellsto the target of the DBDpp (e.g., there is limited to no response whenthe target is not present). Thus, as above, these data indicateDBDpp-CAR T cells targeting PD-L1, proliferate specifically in responseto binding PD-L1. As discussed above, CD137, CD123, and PD-L1 are merelynon-limiting examples of the targets that DBDpp can specifically bindand thus, can (in conjunction with a CAR in a T cell, NK cell, etc.)induce target-specific immune cell function.

Example 11. DBDpp-CAR Transduced T Cells Do Not Display PhenotypesAssociated with T Cell Exhaustion

Persistent exposure of T cells to antigen and/or inflammatory signalscan result in T cell “exhaustion”, characterized by the loss of effectorfunction and expression of multiple inhibitory receptors, such as LAG-3,PD-1 and TIM-3. Such exhaustion can also result from spontaneous T cellstimulation through antigen-independent mechanisms that aggregate T cellreceptors. A consequence of T cell exhaustion can be reduced tumorcontrol, and thus avoidance of excessive exhaustion is a desirableattribute in cancer immunotherapy using T cells.

To assess potential antigen-independent exhaustion in T cells expressingDBDpp-CARs, transduced T-cells (day 10 post-activation) were stainedwith antibodies against CD3 and markers of T-cell exhaustion (LAG3, PD1,and TIM3). FIG. 23A summarizes data from individual experiments acrossseveral T cell donors. The data demonstrate that expression of theexhaustion markers was not enhanced in various CD123-binding DBDpp-CAR Tcells. FIG. 23B shows representative flow cytometry data of LAG-3, PD1,and TIM-3 expression in T-cells transduced with either a scFv-containingCAR (top row) or a DBDpp-CAR (in this particular experiment CD123targeting cg06) 10 days after the initial activation of the T cells. Thesimilarity of these data again demonstrate that DBDpp-CAR T cells do notupregulate expression of exhaustion markers, which lends further supportto their efficacy in cancer immunotherapy.

Example 12. DBDpp-CAR Expressing T Cells Exhibit Target-specificDegranulation and Tumor Cytotoxicity

Degranulation of T cells, NK cells, and many monocytic lineage cells(all of which can be used depending on the embodiment). Degranulationcan result in the release of, depending on the cell type, antimicrobial,cytotoxic or other molecules from secretory granules in the immune cell.Molecules like perforin (a pore forming cytotoxin) or granzymes (serineproteases that induce apoptosis in the target cell) aid T cells and NKcells in killing tumor cells (or other cell types).

To assess degranulation of T cells expressing DBDpp-CARs, 1×10⁵transduced T cells (day 9 post-activation) were cultured in T cell mediafor 4 hours in the presence of monensin and PE-conjugated CD107a/LAMP1.T-cells were cultured alone or in the presence of 2×10⁵ non-target tumorcells (K562, which are CD123-) or target-expressing tumor cells (BDCM,CD123+), then washed and stained for CD3 expression. T-celldegranulation was then assessed by flow cytometry, first gating on theCD3+SSC-low cells (non-tumor), then the CD3+CD107a+ cells. Symbolsrepresent samples from individual experiments using multiple donors.

FIGS. 24A-24D summarize these data. FIG. 24A shows production of CD107a(as a marker of degranulation of the DBDpp-CAR T cells) equivalent tonegative controls when CD123-targeting DBDpp-CAR T cells are culturedalone. FIG. 24B shows limited CD107a expression when DBDpp-CAR T cellsare co-cultured with CD123 negative K562 tumor cells. FIG. 24C showssignificant CD107a expression when CD123-targeting DBDpp-CAR T cells areco-cultured with CD123 positive BDCM cells, thus indicating that the Tcells are activated, undergoing signaling, and undergoing degranulationwill result in tumor eradication. FIG. 24D depicts data fromexperimental replicates of co-culture of CD123-targeting DBDpp-CAR Twith CD123 positive BDCM cells. FIGS. 25A-25D show similar data relatedto degranulation of T cells expressing PD-L1-DBDpp-CARs. Not only dothese data demonstrate that DBDpp-CAR T cells effectively degranulate,these data provide further support for the target-dependent activationof DBDpp-CAR expressing T cells.

Example 13. DBDpp-CAR Mediated Tumor Cytotoxicity is Target Specific

The target-specific function of DBDpp-CAR expressing human T cells wasextended to include in vitro tumor cell cytotoxicity as described inFIGS. 26 and 27. These experiments employed CD123+(BDCM, acutemyelogenous leukemia) and CD123-(K562, chronic myelogenous leukemia)tumor cells that were pre-loaded with fluorescence enhancing ligand(BATDA). CD123-directed DBDpp-CAR expressing T cells were cultured withtumor cells for 2 hours at various effector to target (E:T) ratios. Mockco-culture and co-culture with CD123-directed scFv-CARs were used ascontrols. In an additional group of experiments, PD-L1+(SU-DHL-1, largecell lymphoma) and PD-L1-(K562, chronic myelogenous leukemia) tumorcells were pre-loaded with BATDA. PD-L1-directed DBDpp-CAR expressing Tcells were cultured with tumor cells for 2 hours at various E:T ratios.Mock co-cultures were used as controls. BATDA ligand is released as aresult of the cytolysis of target cells, and upon addition of Europiumsolution (Eu), forms a fluorescent and stable chelate, which wasmeasured using a Synergy 2 (Biotek) time-resolved fluorimeter.

FIGS. 26A-26D and 27A-27F summarize data from these experiments. FIG.26A shows that co-culture of CD123 targeting DBDpp-CAR T cells with K562cells (no CD123 expression) yields a kill percentage less than that ofmock co-culture controls. In contrast FIG. 26B demonstrates that each ofthe groups of T cells expressing CD123 targeting DBDpp-CAR kill CD123positive tumor cells more effectively than mock co-culture controls,and, for some DBDpp, as compared to T cells targeted to CD123 with anscFv. FIGS. 26C and 26D depict similar data with cells from a separatedonor.

As a further example, FIGS. 27A-27F show the kill percentage forPD-L1-directed DBDpp-CAR T cells. As discussed above, when co-culturedwith cells not expressing PD-L1, there is limited or no cytotoxicitydetected (27A, 27C, 27E). However, when co-cultured with cells that doexpress the target marker PD-L1, there is cytotoxicity that is measuredand far exceeds that detected in mock co-culture controls (27B, 27D,27F). These data extend the functional attributes of DBDpp expressing Tcells to include target-specific tumor cell kill.

Example 14. DBDpp Domains Can Be Deimmunized and Retain Function

Many therapeutics have the potential to cause adverse side effects,while providing an effective therapy. In some cases, patients may havean immune reaction to a therapeutic (whether drug or cell based).Because the DBDpp disclosed herein are non-human proteins, in silicoanalyses were performed to identify potentially immunogenic epitopes andeliminate them without compromising the functional properties of theDBDpp.

In silico analysis of the amino acid sequence of cg06 (SEQ ID NO: 99)identified three 9 amino acid sequences that share characteristics withthat of high affinity (binding threshold less than 6%) and promiscuous(present in greater than 50% of relevant alleles) T cell epitopes(Singh, Bioinformatics 17:1236-1237, 2012). Specific amino acidsubstitutions within cg06 were identified as reducing the number ofpredicted T cell epitopes. The corresponding point mutations wereintroduced into cg06, either individually or combination, resulting in aseries of ‘deimmunized’ DBDpp-CARs.

A three-dimensional model of a DBDpp (cg06) is shown in FIG. 28A. FIG.28B depicts cg06 with one (of three) of the potentially immunogenicepitopes modified to be less potentially immunogenic. FIG. 28C depictscg06 with two (of three) of the potentially immunogenic epitopesmodified. FIG. 28D depicts cg06 with all three of the potentiallyimmunogenic epitopes modified.

Jurkat reporter cells were electroporated with these deimmunizedDBDpp-CARs. After 24 hours in culture CAR expression on Jurkat cells wasassessed by staining with anti-FLAG monoclonal antibody. The cells wereco-cultured for 6 hours with CD123+ target tumor cells (KG1a with lowlevel of CD123 expression and BDCM with high level of CD123 expression).NFAT mediated signaling was measured through the addition to the cellsof luciferase assay reagent (Promega) and quantitation of relativeluminescence units (RLU).

Data from these experiments are shown in FIGS. 29A-29B. FIG. 29Ademonstrates that deimmunized CD123-binding DBDpp-CARs (cg06-1 throughcg06-6) induce NFAT signaling when co-cultured with CD123-positive BDCMcells. FIG. 29B further demonstrates that CD123-binding DBDpp-CARs alsoinduce NFAT signaling when co-cultured with cells having lower CD123expression (KG1a)—note the reduced RLU levels compared to FIG. 29A.Taken together, these data demonstrate that the immunogenic potential ofa target-binding DBDpp can be decreased by specific amino acid residuesubstitutions in the DBDpp that do not alter the function of the DBDppor the target density-dependence of CAR-mediated intracellularsignaling—the greater the degree of target present, the greater thedegree of response. These data also indicate that DBDpp according toseveral embodiments disclosed herein can be used as therapeutics and ifneeded, be modified to reduce the potential for immune responses againstthe DBDpp-CAR containing cells (e.g., T cells, NK cells, etc.).

Example 15. Bi-Specific DBDpp

In some instances, target cells (e.g., tumor cells) may express morethan one marker. In some embodiments, the DBD-CARs target a singlemarker (e.g., a unique cancer cell marker) on a target cell. In someembodiments, however, certain markers are not unique to cancer cells,but are also expressed on normal cells (although perhaps at differentlevels). Thus, in several embodiments, targeting two markers presents anopportunity to increase the specificity of an immunotherapeutic agent byengineering a CAR to express a bi-specific DBDpp. In such embodimentsthe targeted cells most efficiently killed would be those expressingboth the markers targeted by the DBDpp (though killing of cellsexpression one or the other of the markers may still occur). To testthis approach, bi-specific DBDpp-CARs were expressed on Jurkat cells andintracellular signaling in response to tumor cells expression one orboth targets was measured.

FIGS. 30A-30B define the cell surface expression of CD123 and PD-L1respectively on K562 (CML); KG-1a (AML); BDCM (AML); SU-DHL-1 (LCL) andH460 (Lung carcinoma) cell lines. K562 does not express either targetwhile KG1a and BDCM express CD123 and low levels of PD-L1 relative tothe high PD-L1 expression observed on the CD123-negative cells, SU-DHLand H460. These cell lines afford the opportunity to determine if theintracellular signaling of a CD123-binding DBDpp-CAR (cg06) can beenhanced by a bi-specific CAR comprising cg06 (anti-CD123) fused tosecond DBDpp with specificity for PD-L1 (pb04). FIG. 31A demonstratesthat DBDpp-CARs comprising cg06 only, (FIG. 31A), pb04 only (FIG. 31B),cg06 fused to the N-terminus of pb04 (cg06-pb04, FIG. 31C), and pb04fused to the N-terminus of cg06 9pb-04-cg06, FIG. 31D) can be transducedand expressed in the Jurkat NFAT reporter cell line as assessed byanti-FLAG mAb binding to the CARs. The ability of the mono-specific andbi-specific CARs to activate the NFAT pathway was assessed byco-culturing the various CARs with tumor cells with different level ofCD123 and/or PD-L1 expression. Cells were co-cultured with target cellsfor 6 hours. NFAT mediated signaling was measured through the additionto the cells of luciferase assay reagent (Promega) and quantitation ofrelative luminescence units (RLU) as a measure of induced intracellularsignaling.

FIG. 31E depicts the results of this experiment. The leftmost group ofbars and the histogram show the relative kill effect of the cg06 DBDppagainst various cell types. Signaling response after co-culture withhighly CD123+ BDCM was the greatest with this DBDpp-CAR. The next groupto the right depicts data showing intracellular signaling afterco-culture of the pb04 DBDpp against the same cell types. Signaling washighest in BDCM, followed by SUHDL1, and H460 (referring to FIGS.30A-30B, these are the highest expressing cell lines for CD123 andPD-L1). The next group to the right depicts data showing intracellularsignaling of a bi-specific cg06-pb04 DBDpp (cg06 more distal to the Tcell membrane as compared to pb04). Finally, the rightmost group showsintracellular signaling from a second bi-specific DBPpp (pb04-cg06DBDpp, where pb04 is more distal to the T cell membrane as compared tocg06). These two groups indicate that bi-specific DBDpp-CARs do functionto promote intracellular signaling. In accordance with severalembodiments, bi-specific DBDpp-CARs show enhanced activity (themagnitude of intracellular signaling in the pb04-cg06 group with BDCMcells is greater than can be accounted for by just the pb04 DBDppalone). Thus in several embodiments, DBDpp-CARs comprising two DBDppscan cooperate to enhance T cell function. In several embodiments, thereis a synergy between the various DBDpp used in a bispecific (or othermultimeric) DBDpp-CAR.

Example 16. DBDpp-Mediated Tumor Immunotherapy In Vivo

In several embodiments, DBDpp-CAR expressing cells are effective atgenerating tumor cytotoxicity in vivo. Experiments will be performed inwhich cancer marker specific DBDpp-CAR are expressed on the surface of Tcells (or in other experiments NK cells). A mouse model will be used, inwhich the mice are genetically engineered to express a solid tumor, orsuspension tumor, in which the tumor cells express the cancer marker towhich the specific DBDpp-CARs are directed. Control mice with tumor thatdoes not express the targeted marker will be used as a control, as willmice receiving a placebo T-cell therapeutic.

The DBDpp-CAR cells will be administered to the mice and tumor burdenwill be assessed over time for the mice receiving various DBDpp-CAR.Tumor burden will be assessed by established methods (e.g., in vivoimaging) and mortality will be assessed over time.

As a result of receiving target specific DBD-pp-CAR cells, tumor burdenwill be reduced in mice expressing the marker specifically targeted bythe DBDpp. The reduction will be significant in comparison to those micereceiving a placebo. Likewise, the reduction will be significant incomparison to those mice having tumor cells that do not express themarker specifically targeted by the DBDpp. Further, mortality will bereduced in the mice receiving the target specific DBDpp-CARs as comparedto the placebo group and the group of mice with tumors not expressingthe specific target marker. The results of this experiment willdemonstrate that immune cells expressing DBDpp-CARs are effectivetherapeutic agents and generate target specific cytotoxicity of tumorcells in vivo.

Example 17. DBDpp-Mediated Tumor Immunotherapy In Vivo

As discussed above, DBDpp-CAR expressing cells are effective atgenerating tumor cytotoxicity in vivo. Experiments will be performed inwhich cancer marker specific DBDpp-CAR are expressed on the surface of Tcells (or in other experiments NK cells). Clinical trials willdemonstrate safety of the DBDpp-CAR cells. Additional trials will beperformed to administer DBDpp-CAR cells to humans having a tumorexpressing a marker, to which the DBDpp is specifically targeted.

The DBDpp-CAR cells will be administered on a schedule to be determinedaccording to ordinary skill in the art. Tumor progression, tumor burdenand mortality will be assessed over time.

As a result of receiving target specific DBDpp-CAR cells, tumorprogression will be slowed and overall tumor burden will be reduced. Thereduction will be significant in comparison historical data utilizingconventional anti-cancer techniques such as chemotherapy or radiationtherapy. Mortality will also decrease in comparison with such therapies.There will be limited off-target cytotoxic effects. The results of thistrial will demonstrate that immune cells expressing DBDpp-CARs areeffective therapeutic agents and generate target specific cytotoxicityof tumor cells in vivo.

Example 18. Competitive Inhibition Assay to Define Target EpitopeSpecificity of DBDpp Comprising Distinct Amino Acid Sequences

DBDpp can be defined by various structural and functional propertiesincluding, but not limited to, primary amino acid sequence, pI, meltingpoint, target-specificity, binding affinity, and target epitopespecificity. The target epitope specificity of a first DBDpp can becompared to that of a second DBDpp using a competitive assay format inwhich the binding of a fixed concentration of the first DBDpp to targetis performed in the presence of increasing concentrations of the secondDBDpp. If the first and second DBDpp bind to the same epitope, or apartially overlapping epitope, as defined by amino acid sequence orspatially, then the second DBDpp will inhibit (e.g., compete for)binding of the first DBDpp to the target. If, however the second DBDppdoes not inhibit the binding of the first DBDpp, then the DBDpp bind todistinct epitopes. This assay format was used to assess the ability of aPD-L1 binding DBDpp, pb04, to inhibit the binding of a fixedconcentration (11.1 pmoles/well) of a second PD-L1 binding DBDpp, pb06.FIG. 32 demonstrates a concentration-dependent inhibition of solubleFLAG-tagged pb06 binding to FC-PD-L1 by soluble pb04 with an IC50concentration of pb04 of 19 pmoles/well. Thus, pb04 and pb06 displayshared epitope binding even though their primary amino acid sequencesdiffer (SEQ ID NO:182 and 184). This assay format is readily adapted tocharacterize the ability of a DBDpp to inhibit ligand binding to thetarget of the DBDpp.

Example 19. Generation and Selection of CD123-Targeting DBDpp

In accordance with several embodiments of the methods disclosed above,the reference sequence of SEQ ID NO: 1 was modified at a plurality ofpositions. In one experiment, the modifications resulted in a library ofDBDpp corresponding to SEQ ID NO: 6. Non-limiting examples of DBDppexhibiting specificity for CD123 are represented by the sequences of SEQID NOS: 60-69.

in an additional experiment, the modifications resulted in a library ofDBDpp corresponding to SEQ ID NO: 2. Non-limiting examples of DBDppexhibiting specificity for CD123 are represented by the sequences of SEQID NOS: 70-91.

In an additional experiment, the modifications resulted in a library ofDBDpp corresponding to SEQ ID NO: 4. Non-limiting examples of DBDppexhibiting specificity for CD123 are represented by the sequences of SEQID NOS: 92-127.

Within any of the libraries generated according to the methods disclosedherein, any of the X_(n) positions in a library sequences can besubstituted with a natural or non-natural amino acid, depending on theembodiment. In some embodiments cysteine and/or proline are not used forsuch substitutions.

Example 20. Generation and Selection of De-Immunized CD123-TargetingDBDpp

In accordance with several embodiments of the methods disclosed above,the reference sequence of SEQ ID NO: 1 was modified at a plurality ofpositions. In one experiment, the modifications resulted in a library ofDBDpp corresponding to SEQ ID NO: 4. According to the methods disclosedherein, select library members were identified and de-immunized byidentifying and modifying potentially immunogenic residues. In thisexperiment, the DBDpp of SEQ ID NO: 99 was modified with an S65Esubstitution to yield SEQ ID NO: 130, exhibiting reduced immunogenicity.

Example 21. De-Immunization of CD123-Targeting DBDpp

A DBDpp of SEQ ID NO: 99 was generated and identified according to themethods disclosed herein. Further modifications were made to reduce thepotential immunogenicity of the DBDpp. In this experiment, a R17Qsubstitution was made to yield the DBDpp of SEQ ID NO: 128.Additionally, a S24E substitution was made to yield the DBDpp of SEQ IDNO: 129. Also, in accordance with several embodiments, multiplede-immunizing substitutions can be made. For example, SEQ ID NO: 99 wasmodified with (i) an R17Q, S24E substitution to yield the DBDpp of SEQID NO: 131, (ii) an R17Q, S24T substitution to yield the DBDpp of SEQ IDNO: 132, (iii) an R17Q, S24G substitution to yield the DBDpp of SEQ IDNO: 133, (iv) an R17Q, S24E, S65E substitution to yield the DBDpp of SEQID NO: 134, (v) an R17Q, S24T, S65E substitution to yield the DBDpp ofSEQ ID NO: 135, and an R17Q, S24G, S65E substitution to yield the DBDppof SEQ ID NO: 136.

Example 22. Generation and Selection of CD19-Targeting DBDpp

In accordance with several embodiments of the methods disclosed above,the reference sequence of SEQ ID NO: 1 was modified at a plurality ofpositions. In one experiment, the modifications resulted in a library ofDBDpp corresponding to SEQ ID NO: 6. Non-limiting examples of DBDppexhibiting specificity for CD19 are represented by the sequence of SEQID NO: 137.

In an additional experiment, the modifications resulted in a library ofDBDpp corresponding to SEQ ID NO: 4. Non-limiting examples of DBDppexhibiting specificity for CD19 are represented by the sequences of SEQID NOS: 138-166.

Within any of the libraries generated according to the methods disclosedherein, any of the X_(n) positions in a library sequences can besubstituted with a natural or non-natural amino acid, depending on theembodiment. In some embodiments cysteine and/or proline are not used forsuch substitutions.

Example 23. Generation and Selection of CD22-Targeting DBDpp

In accordance with several embodiments of the methods disclosed above,the reference sequence of SEQ ID NO: 1 was modified at a plurality ofpositions. In one experiment, the modifications resulted in a library ofDBDpp corresponding to SEQ ID NO: 2. Non-limiting examples of DBDppexhibiting specificity for CD22 are represented by the sequences of SEQID NOS: 167-168.

In an additional experiment, the modifications resulted in a library ofDBDpp corresponding to SEQ ID NO: 4. Non-limiting examples of DBDppexhibiting specificity for CD22 are represented by the sequences of SEQID NOS: 169-176.

Within any of the libraries generated according to the methods disclosedherein, any of the X_(n) positions in a library sequences can besubstituted with a natural or non-natural amino acid, depending on theembodiment. In some embodiments cysteine and/or proline are not used forsuch substitutions.

Example 24. Generation and Selection of DR5-Targeting DBDpp

In accordance with several embodiments of the methods disclosed above,the reference sequence of SEQ ID NO: 1 was modified at a plurality ofpositions. In one experiment, the modifications resulted in a library ofDBDpp corresponding to SEQ ID NO: 6. Non-limiting examples of DBDppexhibiting specificity for DR5 are represented by the sequences of SEQID NOS: 177-178.

In an additional experiment, the modifications resulted in a library ofDBDpp corresponding to SEQ ID NO: 2. Non-limiting examples of DBDppexhibiting specificity for DR5 are represented by the sequence of SEQ IDNO: 179.

In an additional experiment, the modifications resulted in a library ofDBDpp corresponding to SEQ ID NO: 4. Non-limiting examples of DBDppexhibiting specificity for DR5 are represented by the sequence of SEQ IDNO: 180.

Within any of the libraries generated according to the methods disclosedherein, any of the X_(n) positions in a library sequences can besubstituted with a natural or non-natural amino acid, depending on theembodiment. In some embodiments cysteine and/or proline are not used forsuch substitutions.

Example 25. Generation and Selection of PD-L1-Targeting DBDpp

In accordance with several embodiments of the methods disclosed above,the reference sequence of SEQ ID NO: 1 was modified at a plurality ofpositions. In one experiment, the modifications resulted in a library ofDBDpp corresponding to SEQ ID NO: 4. Non-limiting examples of DBDppexhibiting specificity for PD-L1 are represented by the sequences of SEQID NOS: 181-186.

Within any of the libraries generated according to the methods disclosedherein, any of the X_(n) positions in a library sequences can besubstituted with a natural or non-natural amino acid, depending on theembodiment. In some embodiments cysteine and/or proline are not used forsuch substitutions.

All publications, patents, patent applications, internet sites, andaccession numbers/database sequences (including both polynucleotide andpolypeptide sequences) cited are herein incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication, patent, patent application, internet site, or accessionnumber/database sequence were specifically and individually indicated tobe so incorporated by reference.

It is contemplated that various combinations or subcombinations of thespecific features and aspects of the embodiments disclosed above may bemade and still fall within one or more of the inventions. Further, thedisclosure herein of any particular feature, aspect, method, property,characteristic, quality, attribute, element, or the like in connectionwith an embodiment can be used in all other embodiments set forthherein. Accordingly, it should be understood that various features andaspects of the disclosed embodiments can be combined with or substitutedfor one another in order to form varying modes of the disclosedinventions. Thus, it is intended that the scope of the presentinventions herein disclosed should not be limited by the particulardisclosed embodiments described above. Moreover, while the invention issusceptible to various modifications, and alternative forms, specificexamples thereof have been shown in the drawings and are hereindescribed in detail. It should be understood, however, that theinvention is not to be limited to the particular forms or methodsdisclosed, but to the contrary, the invention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the various embodiments described and the appended claims.Any methods disclosed herein need not be performed in the order recited.The methods disclosed herein include certain actions taken by apractitioner; however, they can also include any third-party instructionof those actions, either expressly or by implication. For example,actions such as “administering a T cell comprising a DBDpp-CAR” include“instructing the administration of a T cell comprising a DBDpp-CAR.” Inaddition, where features or aspects of the disclosure are described interms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

The ranges disclosed herein also encompass any and all overlap,sub-ranges, and combinations thereof. Language such as “up to,” “atleast,” “greater than,” “less than,” “between,” and the like includesthe number recited. Numbers preceded by a term such as “about” or“approximately” include the recited numbers. For example, “about 10nanometers” includes “10 nanometers.”

What is claimed is:
 1. A de novo binding domain polypeptide (DBDpp),wherein a) the DBDpp comprises three anti-parallel alpha helices joinedby linker peptides; b) the DBDpp comprises the amino acid sequence ofSEQ ID NO:1 comprising 1 to 30 substitutions selected from the groupconsisting of substitutions of residues 5, 6, 8-10, 12, 13, 15-17,19-27, 29, 30, 32-34, 36, 37, 39-41, 43-52, 54, 55, 57-59, 61, 62,64-66, and 68 of SEQ ID NO: 1, substitution of residues 22-24 of SEQ IDNO: 1 with Z1, and substitution of residues 46-49 of SEQ ID NO: 1 withZ2, wherein Z1 and Z2 independently comprise 2 to 30 amino acids; c) theDBDpp does not comprise the amino acid sequence of SEQ ID NO:50; and d)the DBDpp specifically binds a target of interest.
 2. The DBDpp of claim1, wherein the 1 to 30 substitutions are selected from the groupconsisting of substitutions at residues 5, 6, 8-10, 12, 13, 15-17,19-27, 29, 30, 32-34, 36, 37, 39-41, 43-52, 54, 55, 57-59, 61, 62,64-66, and 68 of SEQ ID NO:
 1. 3. The DBDpp of claim 1, wherein thesubstitutions do not include a substitution with a cysteine or aproline.
 4. The DBDpp of claim 1, wherein the target of interestspecifically bound by the polypeptide is a cancer antigen.
 5. The DBDppof claim 4, wherein the cancer antigen is PD-L1, CD137, or CD123.
 6. TheDBDpp of claim 1, wherein the DBD immunospecifically binds to a proteincomprising amino acids 19-305 of CD123 (SEQ ID NO: 187).
 7. An isolatedpolypeptide comprising the DBDpp of claim
 1. 8. A fusion proteincomprising two of more DBDpp according to claim 1, wherein the aminoacid sequence of the two of more DBDpp comprise identical or differentamino acid sequences.
 9. A composition comprising the polypeptide ofclaim 7 conjugated with a label.
 10. The composition of claim 9, whereinthe label is a biotin moiety, an enzymatic label, a fluorescent label, aluminescent label, or a bioluminescent label.
 11. A compositioncomprising the polypeptide of claim 7 conjugated to a therapeutic orcytotoxic agent.
 12. A composition comprising the polypeptide of claim 7and a pharmaceutically acceptable carrier.
 13. An isolated nucleic acidmolecule encoding the polypeptide of claim
 7. 14. A host cell comprisingthe nucleic acid molecule of claim
 13. 15. A chimeric antigen receptor(CAR), wherein the CAR comprises a target binding domain, atransmembrane domain, and an intracellular signaling domain, wherein thetarget binding domain comprises the DBDpp of claim
 1. 16. The CAR ofclaim 15, wherein the intracellular signaling domain is selected fromthe group consisting of a human CD3 zeta domain, 41BB domain, a CD28domain, or a combination thereof.
 17. The CAR of claim 15, wherein theintracellular signaling domain comprises a costimulatory signalingregion.
 18. An isolated nucleic acid molecule comprising a sequenceencoding the CAR of claim
 15. 19. A cell comprising the nucleic acidmolecule of claim
 18. 20. The cell of claim 19, wherein the DBDpp bindsto a tumor antigen associated with a hematologic malignancy or a solidtumor.
 21. The cell of claim 20, wherein the tumor antigen is selectedfrom the group consisting of CD137, PD-L1, CD123, CTLA4, CD47, KIR, DR5,TIM3, PD1, EGFR, TCR, CD19, CD20, CD22, ROR 1, mesothelin, CD33/1L3Ra,cMet, PSMA, Glycolipid F77, EGFRvIII, GD2, NY-ESO-1, MAGE A3, or acombination thereof.
 22. The cell of claim 19, wherein the cell is a Tcell or a natural killer (NK) cell.
 23. A method of treating a subjecthaving cancer, the method comprising administering to the subject animmune cell comprising a CAR of claim 15, wherein the target bindingdomain specifically binds a target of interest expressed by a cancercell.
 24. The method of claim 23, wherein the immune cell is a T cell ora NK cell.
 25. The method of claim 23, a) wherein the transmembranedomain comprises 41BB or CD28, wherein the cytoplasmic domain comprisesan alpha, beta, or zeta chain of the T cell receptor, and wherein theimmune cell is a T cell, or b) wherein the transmembrane domaincomprises CD28, wherein the cytoplasmic domain comprises a zeta chain ofthe T cell receptor, and wherein the immune cell is a NK cell.
 26. Themethod of claim 23, wherein the target binding domain binds to a tumorantigen associated with a hematologic malignancy or a solid tumor. 27.The method of claim 26, wherein the tumor antigen is selected from thegroup consisting of CD137, PD-L1, CD123, CTLA4, CD47, KIR, DR5, TIM3,PD1, EGFR, TCR, CD19, CD20, CD22, ROR 1, mesothelin, CD33/1L3Ra, cMet,PSMA, Glycolipid F77, EGFRvIII, GD2, NY-ESO-1, MAGE A3, or a combinationthereof.
 28. The method of claim 23, wherein the CAR further comprises asecond target binding domain having a different target than the targetbinding domain.